Substrate trapping protein tyrosine phosphatases

ABSTRACT

Novel protein tyrosine phosphatases in which the invariant aspartate residue is replaced with an alanine residue and which bind to a tyrosine phosphorylated substrate and are catalytically attenuated are described. Also described are methods of identifying tyrosine phosphorylated proteins which complex with the described protein tyrosine phosphatases.

FUNDING

The invention was supported, in whole or in part, by grant 5-RO1-CA53840-05 from the National Institutes of Health. The U.S. Government has certain rights in the invention.

RELATED APPLICATIONS

This application is a Divisional under 37 C.F.R. §1.53(b) of prior U.S. application Ser. No. 08/685,992, filed Jul. 25, 1996, the contents of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

The protein tyrosine phosphatase (PTP) family of enzymes consists of more than 500 structurally diverse proteins which have in common the highly conserved 250 amino acid PTP catalytic domain, but which display considerable variation in their non-catalytic segments (Charbonneau and Tonks, Annu. Rev. Cell Biol. 8:463-493 (1992); Tonks, Semin. Cell Biol. 4:373-453 (1993)). This structural diversity presumably reflects the diversity of physiological roles of individual PTP family members, which in certain cases have been demonstrated to have specific functions in growth, development and differentiation (Desai et al., Cell 84:599-609 (1996); Kishihara et al., Cell 74:143-156 (1993); Perkins et al., Cell 70:225-236 (1992); Pingel and Thomas, Cell 58:1055-1065 (1989); Schultz et al., Cell 73:1445-1454 (1993)). Although recent studies have also generated considerable information regarding the structure, expression and regulation of PTPs, the nature of the tyrosine phosphorylated substrates through which the PTPs exert their effects remains to be determined. Studies with a limited number of synthetic phosphopeptide substrates have demonstrated some differences in substrate selectivity of different PTPs (Cho et al., Protein Sci. 2: 977-984 (1993); Dechert et al., Eur. J. Biochem. 231:673-681 (1995)), and have indicated preferences for certain amino acid residues at particular positions around the phosphorylated tyrosine residue (Ruzzene et al., Eur. J. Biochem. 211:289-295 (1993); Zhang et al., Biochemistry 33:2285-2290 (1994)). This indicates that PTPs display a certain level of substrate selectivity in vitro, although the physiological relevance of the substrates used in these studies is unclear.

SUMMARY OF THE INVENTION

As described herein, the substrate specificity of mammalian protein tyrosine phosphatases (PTPs) has been investigated using a novel substrate trapping approach in which mutant or altered forms of the mammalian PTP, also referred to as substrate trapping PTPs, are used to bind (trap) one or more substrates of the PTP. Binding of the substrate trapping PTP with a substrate of the PTP results in the formation of a complex which can be readily observed, and, if desired, isolated, and characterized. The mutant forms of the PTPs have attenuated catalytic activity (lack catalytic activity or have reduced catalytic activity) relative to the wild type PTP but retain the ability to bind tyrosine phosphorylated substrate(s) of the wild type PTP.

The methods of the present invention are specifically exemplified herein with respect to the phosphatases PTP1B and PTP-PEST; however, it is understood that the invention is not limited to these specific PTPs but is applicable to all members of the PTP family. In order to identify potential substrates of PTP1B and PTP-PEST, mutant (i.e., altered or substrate trapping) forms of PTP1B and PTP-PEST were generated which were catalytically attenuated but retained the ability to bind substrates. These mutant PTPs associated in stable complexes with proteins which were identified by immunoblotting as p210 bcr:ab1 and p₁₃₀ ^(cas), respectively. These associations were observed in lysates from several cell lines and in transfected COS cells, indicating that p210 bcr:ab1 and p130^(cas) represent major physiologically relevant substrates for PTP1B and PTP-PEST.

These results provide the first demonstration of PTPs having inherently restricted substrate specificity in vivo. The methods used to identify p210 bcr:ab1 and p130^(cas) as specific substrates for PTP1B and PTP-PEST, respectively, are generally applicable to any member of the PTP family, of which approximately 500 members have currently been reported, and can be used to determine the physiological substrates of other members of the PTP family.

One embodiment of the invention relates to novel mutant PTPs in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹). These PTPs retain the ability to form a complex with, or bind, their tyrosine phosphorylated substrates, but are catalytically attenuated. In one embodiment, the invention relates to the phosphatase PTP1B in which the invariant aspartate residue at position 181 is replaced with alanine (D181A). In another embodiment the invention relates to the phosphatase PTP-PEST in which the invariant aspartate residue at position 199 is replaced with an alanine (D199A). Another embodiment of the invention relates to a PTP-PEST phosphatase in which the cysteine residue at position 231 is replaced with a serine (C231S). The invention also relates to other mutant or substrate trapping PTPs in which the invariant aspartate residue is replaced with or changed to another amino acid residue, such as alanine. The invariant aspartate residue can be identified in other PTPs by aligning the PTP nucleotide sequence with the nucleotide sequence of a PTP for which the location of the invariant aspartate residue is known.

The invention also relates to a method of identifying a tyrosine phosphorylated substrate of a protein tyrosine phosphatase. According to one embodiment of the present invention, a tyrosine phosphorylated protein of interest is combined with one or more PTP(s) in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹), and the presence or absence of a complex between the protein and the PTP(s) is determined. Presence of a complex in the combination indicates that the tyrosine phosphorylated protein is a substrate of the PTP. The PTP DA mutant binds to or complexes with its substrate but does not dephosphorylate it (or does so very slowly), thereby allowing the complex to be observed and, optionally, isolated and identified. In a particular embodiment of the invention, the invariant aspartate is replaced with an alanine residue (a PTP DA mutation or alteration)

In an alternative embodiment of the present invention, a PTP of interest in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹), is combined with one or more tyrosine phosphorylated proteins, and the presence or absence of a complex between the protein(s) and the PTP is determined. Presence of a complex in the combination indicates that the tyrosine phosphorylated protein is a substrate of the PTP. The PTP DA mutant binds to or complexes with its substrate but does not dephosphorylate it (or does so very slowly), thereby allowing the complex to be observed, and, optionally, isolated and identified. In one embodiment of the invention, the invariant aspartate residue is replaced with an alanine residue (a PTP DA mutation or alteration)

The present invention also relates to a method of identifying a tyrosine phosphorylated substrate of a protein tyrosine phosphatase wherein more than one tyrosine phosphorylated protein of interest is combined with more than one PTP of interest in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹) (e.g., the invariant aspartate is replaced with an alanine residue). Complexes formed in the combination can be isolated and the component PTP and substrate can be identified.

The invention also pertains to a method of reducing the activity of a tyrosine phosphorylated protein, comprising administering to a mammal a PTP in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹) (e.g., the invariant aspartate is replaced with an alanine residue) and which forms a complex with the tyrosine phosphorylated protein. The PTP mutant binds to the phosphorylated protein without dephosphorylating it, thereby inhibiting the activity of the protein and reducing its downstream effects.

For example, the invention relates to a method of reducing the transforming effects of oncogenes associated with p130^(cas), a substrate of PTP-PEST, comprising administering to a mammal wild type PTP-PEST or PTP-PEST in which the invariant aspartate residue is replaced with an alanine residue. Wild type PTP-PEST binds and dephosphorylates p130^(cas), thereby negatively regulating its downstream effects. DA mutants of PTP-PEST bind but do not dephosphorylate p130^(cas) (or dephosphorylate it at a reduced rate); the substrate is thus tied up in the complex with the substrate trapping form of PTP-PEST and cannot exert its downstream effects. Similarly, the invention relates to a method of reducing the formation of signalling complexes associated with p130^(cas), particularly those signalling complexes which induce mitogenic pathways, comprising administering to a mammal wild type PTP-PEST or PTP-PEST in which the invariant aspartate residue is replaced with an alanine residue.

The present invention also relates to assays for identifying agents which alter, e.g., enhance or inhibit, the interaction between a PTP and its phosphorylated substrate. Agents identified by these assays can be agonists (e.g., agents which enhance or increase the activity of the PTP) or antagonists (e.g., agents which inhibit or decrease the activity of the PTP) of PTP activity. The agent may be an endogenous physiological substance or may be a natural or synthetic drug, including small organic molecules.

For example, the tyrosine phosphorylated substrate of a PTP can be identified by the methods described herein. An enzymatic activity assay utilizing the wild type PTP can be carried out in the presence of an agent to be tested, and the resulting amount of enzyme activity can be compared with the amount of enzyme activity in the absence of the agent to be tested. A decrease in the enzymatic activity in the presence of the agent to be tested indicates that the agent inhibits the interaction between the PTP and its substrate. Conversely, an increase in the enzymatic activity in the presence of the agent to be tested indicates that the agent enhances the interaction between the PTP and its substrate.

Alternatively, a competitive binding assay can be carried out utilizing the mutant PTP in the presence of an agent to be tested, and the resulting extent of binding of the mutant PTP to its substrate can be compared with the extent of binding in the absence of the agent to be tested. A decrease in the extent of binding in the presence of the agent to be tested indicates that the agent inhibits the interaction between the PTP and its substrate. Conversely, an increase in the extent of binding in the presence of the agent to be tested indicates that the agent enhances the interaction between the PTP and its substrate.

Thus, the compositions and methods described herein are useful in identifying the tyrosine phosphorylated substrates of members of the PTP family of phosphatases, as well as in regulating the activity of identified substrates. The compositions and methods described herein are also useful for identifying tyrosine phosphorylated proteins which are related to a particular disease or disorder, and to methods of screening for modulators which enhance or inhibit the PTP/substrate interaction for use in therapeutic applications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show a multiple sequence alignment of the catalytic domains of PTPs (SEQ ID NOS. 1-35). Cytosolic eukaryotic PTPs and domain 1 of RPTPs are combined into one group domains 2 of RPTPs are in a second group and the Yersinia PTP is in a third. Invariant residues shared among all three groups are shown in lower case. Invariant and highly conserved residues within a group are shown in italics and bold, respectively. Within the Yersinia PTP sequence, residue that are either invariant or highly conserved between the cytosolic and RPTP domain sequences are italics and bold, respectively. The position of residues of PTP1B that interact with the peptide are indicated with a small arrowhead, and the residue numbering at the bottom of the alignment corresponds to that for PTP1B.

FIG. 2 shows the Vmax and Km of various PTP1B mutants toward RCML.

DETAILED DESCRIPTION OF THE INVENTION

The PTP family of enzymes contains a common evolutionarily conserved segment of approximately 250 amino acids known as the PTP catalytic domain. Within this conserved domain is a unique signature sequence motif, I/V!HCXAGXXR S/T!G (SEQ ID NO: 36), that is invariant among all PTPs. The cysteine residue in this motif is invariant in members of the family and is known to be essential for catalysis. It functions as a nucleophile to attack the phosphate moiety of the incoming substrate. If the cysteine residue is altered by site-directed mutagenesis to serine (CS mutants) or alanine (CA mutants), the resulting PTP is catalytically attenuated but retains the ability to complex with, or bind, its substrate, at least in vitro. These results have been confirmed relative to MKP-1, a member of the PTP family (Sun et al., Cell 75:487-493 (1993)), as well as other PTPs. However, although these CS mutants can in general bind effectively to phosphotyrosyl substrates in vitro, in many cases such complexes cannot be isolated in vivo. Thus, the CS mutants are limited in their applicability and cannot be used to isolate all combinations of PTPs and substrates.

The crystal structures of PTP1B alone (Barford, et al., Science 263:1397-1404 (1994)) and in a complex with a phosphotyrosine-containing peptide (Jia et al., Science 268:1754-1758 (1995)) were recently determined. These structures indicated twenty seven invariant residues (Barford et al., 1994), one of which is an aspartate residue. This aspartate residue is invariant across the catalytic domains of PTP family members. That is, if the amino acid sequences of the PTP family members are aligned, the aspartate residue is present in each PTP at a corresponding location, although the position numbers may be different due to the shifts required to maximize alignment (see FIG. 1 (from Barford et al., Nature Struc. Biol. 2:1043-1053 (1995)) for an alignment of various PTP sequences). Sequences for which the alignment has not yet been published can readily be aligned with other known PTP sequences, e.g., utilizing available computer software such as GENEWORKS.

Thus, mutant PTPs other than those specifically described herein can readily be made by aligning the amino acid sequence of the PTP catalytic domain with those described herein, identifying the invariant aspartate residue, and changing the residue by site-directed mutagenesis. Although the specific examples of PTP mutants described herein are aspartate to alanine mutants (DA mutants), it is understood that the invention is not limited to changes of aspartate to alanine. The invariant aspartate residue can be changed, e.g., by site-directed mutagenesis, to any amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹). For example, the invariant aspartate residue can be changed or mutated to an alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine or histidine.

As described herein, pervanadate-treated cells were used as an abundant source of tyrosine phosphorylated proteins to investigate the substrate specificity of PTP-PEST. PTP-PEST is an 88 kDa cytosolic PTP (Charest et al., Biochem. J. 308:425-432 (1995); den Hertog et al., Biochem. Biophys. Res. Commun. 184:1241-1249 (1992); Takekawa et al., Biochem. Biophys. Res. Commun. 189:1223-1230 (1992); Yang et al., J. Biol. Chem. 268:6622-6628 (1993); Yang et al., J. Biol. Chem. 268:17650 (1993)) which is expressed ubiquitously in mammalian tissues (Yi et al., Blood 78: 2222-2228 (1991)), and which exhibits high specific activity when assayed in vitro using artificial tyrosine phosphorylated substrates (Garton and Tonks, EMBO J. 13:3763-3771 (1994)). It has previously been demonstrated that PTP-PEST is subject to regulation via phosphorylation of Ser39 in vitro and in vivo. This modification is catalyzed by both protein kinase C (PKC) and protein kinase A (PKA), and results in reduced enzyme activity as a consequence of an increase in the Km of the dephosphorylation reaction (Garton and Tonks, EMBO J. 13:3763-3771 (1994)). It appears likely that further regulatory mechanisms exist for PTP-PEST, since this enzyme would be expected to exert a considerable negative influence on the tyrosine phosphorylation state of cytosolic substrates of tyrosine kinases. One possibility is that this influence could be limited by the substrate specificity of PTP-PEST.

The substrate specificity of PTP 1B was investigated utilizing the same methods outlined for PTP-PEST, with the exception that the cells were not treated with pervanadate. A combination of in vitro dephosphorylation and substrate trapping experiments were used to study the substrate interactions of PTP1B and PTP-PEST. The substrate trapping methods outlined herein are generally applicable to any PTP by virtue of the shared invariant aspartate residue, and should therefore prove useful in delineating the substrate preference of other PTP family members. In particular, the use of mutant, catalytically impaired PTPs to trap, and thereby isolate, potential substrates will greatly facilitate the identification of physiologically important substrates for individual PTPs, leading to improved understanding of the roles of these enzymes in regulation of cellular processes.

One embodiment of the invention relates to novel PTPs in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹). These PTPs retain the ability to form a complex with, or bind, their tyrosine phosphorylated substrates but are catalytically attenuated. As defined herein, "attenuated" activity is intended to mean that the phosphatase retains a similar Km to that of the wild type phosphatase but has a Vmax which is reduced by a factor of at least 10⁴ relative to the wild type enzyme. This includes catalytic activity which is either reduced or abolished relative to the wild type PTP. For example, the invariant aspartate residue can be changed or mutated to an alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, lysine, arginine or histidine.

The novel PTPs described herein, in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹), can also comprise other mutations, particularly those which assist in stabilizing the PTP/substrate complex. For example, a mutation of the serine/threonine! residue in the signature motif to an alanine residue changes the rate-determining step of the dephosphorylation reaction from the formation of the transition state to the break down of the transition state, thereby stabilizing the PTP/substrate complex. Such mutations may be valuably combined with the replacement of the invariant aspartate residue, particularly assisting in stabilizing the complex and facilitating the observation and isolation of the complex.

PTPs suitable for use in the invention include any PTP which has an invariant aspartate residue in a corresponding position. As defined herein, a phosphatase is a member of the PTP family if it contains the signature motif I/V!HCXAGXXR S/T!G. Dual specificity PTPs, i.e., PTPs which dephosphorylate both phosphorylated tyrosine and phosphorylated serine or threonine, are also suitable for use in the invention. Appropriate PTPs include, but are not limited to, PTP1B, PTP-PEST, PTP.sub.γ, MKP-1, DEP-1, PTPμ, PTPX1, PTPX10 and PTPH1.

In one embodiment, the invention relates to the phosphatase PTP1B in which the aspartate residue at position 181 is replaced with alanine (D181A). In another embodiment the invention relates to the phosphatase PTP-PEST in which the invariant aspartate residue at position 199 is replaced with an alanine (D199A). Another embodiment of the invention relates to a PTP-PEST phosphatase in which the cysteine residue at position 231 is replaced with a serine (C231S).

The invention also relates to a method of identifying a tyrosine phosphorylated protein which is a substrate of a particular protein tyrosine phosphatase. According to one embodiment of the present invention, a tyrosine phosphorylated protein of interest is combined with at least one PTP in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹) (e.g., an alanine residue), and the presence or absence of a complex between the protein and the PTP is determined. Presence of a complex in the combination indicates that the tyrosine phosphorylated protein is a substrate of the PTP. The PTP DA mutant (substrate trapping mutant) binds to or complexes with its substrate but does not dephosphorylate it (or does so very slowly), thereby allowing the complex to be isolated and identified.

The phosphorylated protein/PTP complex may be isolated by conventional isolation techniques as described in U.S. Pat. No. 5,352,660 to Pawson, including salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof. Furthermore, to facilitate the determination of the presence of the protein/PTP complex, antibodies against the PTP or the phosphorylated protein can be used, as well as labelled PTPs and/or labelled phosphorylated substrates. The PTP or phosphorylated protein can be labelled with various enzymes, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include, but are not limited to, horseradish peroxidase, biotin, alkaline phosphatase, β-galactosidase and acetylcholinesterase. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. Appropriate luminescent materials include luminol, and suitable radioactive material include radioactive phosphorous ³² P, iodine I¹²⁵, I¹³¹ or tritium.

Alternatively, the invention pertains to a method of identifying a tyrosine phosphorylated protein which is a substrate of a PTP, comprising combining a PTP of interest in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹) (e.g., an alanine residue), with at least one tyrosine phosphorylated protein, thereby producing a combination; and determining the presence or absence of a complex in the combination, wherein presence of a complex in the combination between a tyrosine phosphorylated protein and the PTP indicates that the tyrosine phosphorylated protein is a substrate of the PTP.

The substrate trapping PTPs of the present invention can also be used in place of wild type PTPs to screen phosphotyrosyl peptide libraries for peptides which bind to the PTP as described in Songyang et al. (Nature 373:536-539 (1995); Cell 72:767-778 (1993)). Peptides identified from such peptide libraries can then be assessed to determine whether tyrosine phosphorylated proteins containing these peptides exist in nature.

Any tyrosine phosphorylated protein is suitable as a potential substrate in the present invention. Tyrosine phosphorylated proteins are well known in the art. Specific examples of appropriate substrates include, without limitation, p130^(cas), the EGF receptor, p210 bcr:ab1, MAP kinase and the insulin receptor. Of particular interest are tyrosine phosphorylated proteins which have been implicated in a mammalian disease or disorder.

The invention also pertains to a method of reducing the activity of a tyrosine phosphorylated protein, comprising administering to a mammal a PTP in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹) (e.g., an alanine residue) and which forms a complex with the tyrosine phosphorylated protein. The PTP DA mutant binds to the phosphorylated protein without dephosphorylating it (or causing dephsophorylation at a greatly reduced rate), thereby inhibiting the activity of the protein and reducing its downstream effects. As used herein, "reducing" includes both reduction and complete abolishment, e.g., of one or more activities or functions of the phosphorylated protein.

For example, the invention relates to a method of reducing the transforming effects of oncogenes associated with p130^(cas), a substrate of PTP-PEST, comprising administering to a mammal wild type PTP-PEST or PTP-PEST in which the invariant aspartate residue is replaced with an alanine residue. Wild type PTP-PEST binds and dephosphorylates p130^(cas), thereby negatively regulating its downstream effects. DA mutants of PTP-PEST bind but do not dephosphorylate p130^(cas) (or do so at a greatly reduced rate); the substrate is thus tied up in the complex with the substrate trapping form of PTP-PEST and cannot exert its downstream effects. Similarly, the invention relates to a method of reducing the formation of signalling complexes associated with p130^(cas), particularly those signalling complexes which induce mitogenic pathways, comprising administering to a mammal wild type PTP-PEST or PTP-PEST in which the invariant aspartate residue is replaced with an alanine residue. The PTP binds to and/or dephosphorylates p130^(cas), thereby negatively regulating the downstream effects of p130^(cas) and reducing the formation of signalling complexes associated with p130^(cas).

The substrate trapping mutant PTPs of the present invention can be used in virtually any application in place of, or in addition to, a corresponding wild type PTP. The advantages of such a utility lie in the ability of the mutant PTP to mimic the function of the wild type enzyme, e.g., to decrease the activity of its tyrosine phosphorylated substrate, without inducing the harmful cytotoxic effects commonly observed with administration or overexpression of the wild type PTP. Thus, the invention also pertains to a method of reducing the cytotoxic effects associated with administration or overexpression of wild type PTPs. For example, CS mutants of MKP-1 have been shown to have the same functional effect as wild type MKP-1 without induction of potentially harmful side effects. Thus, PTPs described herein, in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹) (e.g., an alanine residue), can be used in many applications in place of the corresponding wild type enzyme. As used herein, a "corresponding" enzyme is one which is the same as the mutant PTP (e.g., PTP-PEST and PTP-PEST D199A) or one which is different from the mutant PTP but recognizes the same substrate as the mutant PTP.

The mutant PTPs described herein can also be used therapeutically to reduce the activity of a tyrosine phosphorylated protein, such as by a gene therapy method in which the mutant PTP described herein, or a functional portion thereof which retains the ability to bind to its tyrosine phosphorylated substrate, is introduced into a subject and in whom the mutant PTP is expressed. The mutant PTP replaces, either partially or totally, the wild type enzyme which is normally produced or competes with the wild type PTP for binding to the substrate. For example, a specific tyrosine phosphorylated protein can be identified which is implicated in a particular disease or disorder (such as a protein tyrosine kinase). At least one PTP which acts to dephosphorylate the selected tyrosine phosphorylated protein of the present invention can be identified by the methods described herein. The wild type or mutant form of the PTP can be administered to a subject in need of treatment in order to tie up or bind the tyrosine phosphorylated substrate, thereby inhibiting or reducing the function of the phosphorylated protein. In a preferred embodiment, the mutant PTP is administered in place of the wild type enzyme in order to reduce the cytotoxic effects associated with overexpression of the wild type enzyme. Procedures for gene therapy are known in the art (see U.S. Pat. No. 5,399,346 to Anderson et al.) and can be modified by methods known in the art to appropriately express the specific mutant and wild type PTPs of the present invention.

The present invention also relates to assays for identifying agents which alter, e.g., enhance or inhibit, the interaction between a PTP and its phosphorylated substrate. Agents identified by these assays can be agonists (e.g., agents which enhance or increase the activity of the PTP) or antagonists (e.g., agents which inhibit or decrease the activity of the PTP) of PTP activity. The agent may be an endogenous physiological substance or may be a natural or synthetic drug, including small organic molecules.

For example, the tyrosine phosphorylated substrate of a PTP can be identified by the methods described herein. An enzymatic activity assay utilizing the wild type PTP can be carried out in the presence of an agent to be tested, and the resulting amount of enzyme activity can be compared with the amount of enzyme activity in the absence of the agent to be tested. Enzymatic activity assays are known in the art; for example, assays of PTP activity using a tyrosine phosphorylated ³² P-labelled substrate are described in Flint et al. (EMBO J. 12:1937-1946 (1993)). A decrease in the enzymatic activity in the presence of the agent to be tested indicates that the agent inhibits the interaction between the PTP and its substrate. Conversely, an increase in the enzymatic activity in the presence of the agent to be tested indicates that the agent enhances the interaction between the PTP and its substrate.

Alternatively, a competitive binding assay can be carried out utilizing the mutant PTP in the presence of an agent to be tested, and the resulting extent of binding of the mutant PTP to its substrate can be compared with the extent of binding in the absence of the agent to be tested. Competitive binding assays are known in the art; for example, U.S. Pat. No. 5,352,660 to Pawson describes methods suitable for use in this invention. A decrease in the extent of binding in the presence of the agent to be tested indicates that the agent inhibits the interaction between the PTP and its substrate. Conversely, an increase in the extent of binding in the presence of the agent to be tested indicates that the agent enhances the interaction between the PTP and its substrate.

According to the present invention, tyrosine phosphorylated peptides identified with mutant PTPs from peptide libraries by the methods of Songyang et al. (Nature 373:536-539 (1995); Cell 72:767-778 (1993)) can be used herein in place of the complete tyrosine phosphorylated protein in competitive binding assays.

The present invention also pertains to pharmaceutical compositions comprising a PTP in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute (less than 1 min⁻¹) (e.g., an alanine residue). For instance, the PTP of the present invention can be formulated with a physiologically acceptable medium to prepare a pharmaceutical composition. The particular physiological medium may include, but is not limited to, water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists, and will depend on the ultimate pharmaceutical formulation desired. Methods of introduction of exogenous PTPs at the site of treatment include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal. Other suitable methods of introduction can also include rechargeable or biodegradable devices and slow release polymeric devices. The pharmaceutical compositions of this invention can also be administered as part of a combinatorial therapy with other agents.

The following Examples are offered for the purpose of illustrating the present invention and are not to be construed to limit the scope of this invention. The teachings of all references cited herein are hereby incorporated by reference in their entirety.

EXAMPLES

Materials and Methods

The following is a description of the materials and methods used in the work described herein.

Generation, Expression and Purification of Mutant PTP Proteins

Point mutations within the catalytic domains of PTP-PEST (D 199A, C231S) and PTP1B (D181A, C215S) were introduced by site-directed mutagenesis using the Muta-Gene™ in vitro mutagenesis kit (Bio-Rad, Richmond, Calif.). Regions containing the required point mutation were then exchanged with the wild type sequences within appropriate expression vectors, and the replaced mutant regions were sequenced in their entirety to verify the absence of additional mutations.

Full length PTP-PEST proteins (wild type and mutant proteins, containing either Asp199 to Ala or Cys231 to Ser mutations) and the wild type PTP-PEST catalytic domain (amino acids 1-305) were expressed in Sf9 cells using recombinant baculovirus (BaculoGold™, Pharmingen, San Diego, Calif.), and purified as described in Garton and Tonks (EMBO J. 13:3763-3771 (1994)). Truncated forms of wild type and mutant PTP-PEST proteins, comprising amino acid residues 1-305 of PTP-PEST were also expressed in E. coli as GST fusion proteins following subcloning of PTP-PEST DNA in-frame downstream of GST in pGEX vectors (Pharnacia Biotech Inc., Uppsala, Sweden). Twenty-five ml of E. coli transformed with the appropriate vector were grown to log phase (OD₆₀₀ approximately 0.5). Fusion protein expression was then induced by addition of 0.2 mM isopropyl-1-thio-b-D-galactopyranoside, and the cells were grown for 2-4 hours at 30° C. Cells were harvested by centrifugation, incubated with 50 mg/ml lysozyme in 3 ml buffer containing 50 mM Tris-HCl, pH 7.4, 5mM EDTA, 1 mM PMSF, 1 mM benzamidine, 5 mg/ml leupeptin, 5 mg/ml aprotinin, 0.1% Triton X-100 and 150 mM NaCl, then lysed by sonication (3×10s). Following removal of insoluble material by centrifugation (20 minutes at 300,000×g), fusion proteins were isolated by incubation for 30 min at 4° C. with 100 ml glutathione-Sepharose beads (Pharmacia Biotech Inc., Uppsala, Sweden), and the beads were then collected by centrifugation and washed three times with buffer A (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM benzamidine, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 10% glycerol, 1% Triton X-100 and 100 mM NaCl). This procedure yielded essentially homogeneous fusion protein at a concentration of 1 mg protein/ml glutathione-Sepharose beads. PTP1B proteins (wild type and mutant forms) comprising amino acids 1-321 were expressed in E.coli and purified to homogeneity as described in Barford et al. (J. Mol. Biol. 239:726-730 (1994)).

Cell Culture, Transfection, Preparation of Lysates and Fractionation

HeLa and COS cells were grown in Dulbecco's modified Eagle's medium (DMEM), containing 5% fetal bovine serum (FBS); Wi38, C2C12 and MvLu cells were grown in DMEM containing 10% FBS; 293 cells were grown in DMEM containing 10% calf serum; MCF10A cells were grown in 50% DMEM, 50% Ham's F-12 containing 5% horse serum, 20 ng/ml epidermal growth factor, 10 mg/ml insulin, 0.5 mg/ml hydrocortisone and 0.25 mg/ml fungizone. All media also contained penicillin and streptomycin at 100 U/ml and 100 mg/ml, respectively, and all cells were grown at 37° C. Calcium phosphate-mediated transfection was used to introduce cDNA encoding wild type and mutant PTP-PEST proteins into COS cells. These were encoded by PTP-PEST cDNA subcloned into the plasmid PMT2, from which expression was driven by an adenovirus major late promoter; 20 mg DNA was used for transfection of each 10 cm plate of cells. The level of expression of PTP-PEST constructs was similar in all cases.

Prior to cell lysis, 70-90% confluent cultures of cells were treated for 30 minutes with 0.1 mM pervanadate (20 ml of a fresh solution containing 50 mM sodium metavanadate (NaVO₃) and 50 mM H₂ O₂ were added to 10 ml medium). Treatment of cells with H₂ 0₂ and vanadate leads to a synergistic increase in phosphotyrosine levels, presumably due to inhibition of intracellular PTPs by vanadate. The synergism between H₂ O₂ and vanadate has previously been suggested to result from improved accumulation of the resultant oxidized vanadate (pervanadate) within the cells when compared to vanadate itself (Heffetz et al., J. Biol. Chem. 265:2896-2902 (1990)). It is important to note that during the preparation of cell lysates, dilution occurs such that the inhibitory effect of vanadate on PTP action is lost. Pervanadate treatment resulted in the appearance of at least 50 prominent phosphotyrosine protein bands in all cell types, whereas untreated cells contained virtually undetectable levels of phosphotyrosine (data not shown).

Cells were lysed in Buffer A containing 5 mM iodoacetic acid, which was included in order to inhibit irreversibly cellular PTPs. Following incubation at 4° C. for 30 minutes, 10 mM DTT was added to inactivate any unreacted iodoacetic acid. Insoluble material was then removed by centrifugation for 20 minutes at 300,000×g. The resultant lysates were stable with regard to their phosphotyrosine content during long term (several months) storage at -70° C. and during prolonged (at least 20 hours) incubation at 4° C., in the absence of exogenous added PTPs.

Pervanadate-treated HeLa cell lysate was fractionated by anion exchange chromatography using a Mono Q FPLC column (Pharmacia). The sample (50 mg total protein at 3 mg/ml in buffer A) was diluted in three volumes of buffer B (20 mM tris-HCl, pH 7.4, 1 mM EDTA, 1 mM benzamidine, 1 mg/ml leupeptin, 1 mg/ml aprotinin and 0.1% Triton X-100) prior to loading. Proteins were eluted at a flow rate of 1 ml/min with a linear gradient of 0-0.5 M NaCl in buffer B over 20 fractions (1 ml fraction volume), followed by a second gradient of 0.5-1.0 M NaCl in buffer B over 5 fractions. Phosphotyrosine-containing proteins were detected within fractions 7-21 according to anti-phosphotyrosine immunoblotting. The same procedures were followed for PTP1B, with the exception that the cells were not treated with pervanadate.

Dephosphorylation Reactions

Lysates of pervanadate-treated HeLa cells (1-2 mg protein/ml) containing tyrosine phosphorylated proteins were incubated on ice in the absence or presence of purified active PTPs at a concentration of 2 nM. Dephosphorylation was terminated by the removal of aliquots (30 mg protein) into SDS-PAGE sample buffer, and the extent of dephosphorylation was determined by immunoblotting using the monoclonal antibody G104. Assays of PTP activity using tyrosine phosphorylated ³² P-labelled RCM-lysozyme as substrate were performed as described in Flint et al. (EMBO J. 12:1937-1946 (1993)).

Antibodies and Immunoblotting

The PTP-PEST monoclonal antibody AG25 was raised against baculovirus-expressed purified full-length PTP-PEST. The anti-phosphotyrosine monoclonal antibody G104 was generated using as antigen phosphotyrosine, alanine and glycine, in a 1:1:1 ratio, polymerized in the presence of keyhole limpet hemocyanin with 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide, a method originally described in Kamps and Sefton (Oncogene 2:305-315 (1988)). p130^(cas) monoclonal antibody was from Transduction Laboratories (Lexington, Ky.). Monoclonal antibody FG6 against PTP1B was provided by Dr David Hill (Calbiochem Oncogene Research Products, Cambridge, Mass.). Visualization of proteins by immunoblotting was achieved by enhanced chemiluminescence (ECL) using HRP-conjugated secondary antibodies (Amersham Life Science Inc., Arlington Heights, Ill.) and the SuperSignal™ CL-HRP substrate system (Pierce, Rockford, Ill.).

Immunoprecipitation and Substrate Trapping

Immunoprecipitation of PTP-PEST from transfected COS cells was performed following covalent coupling of monoclonal antibody AG25 to protein A-Sepharose beads (Pharmacia Biotech Inc., Uppsala, Sweden) using the chemical cross-linking agent dimethyl pimelimidate (Schneider et al., J. Biol. Chem. 257:10766-10769 (1982)). Antibody was first bound to protein A-Sepharose at a concentration of 1 mg/ml bead volume, and unbound material was then removed by three washes with 0.2 M sodium borate, pH 9. Covalent coupling was achieved by incubation at room temperature for 30 minutes in the presence of 20 mM dimethyl pimelimidate in 0.2 M sodium borate, pH 9. The beads were then incubated for 1 hour with an excess of 0.2 M ethanolamine, pH 8, to block any unreacted cross-linker, and washed three times with PBS prior to storage at 4° C. Ten ml of AG25 beads were used to precipitate transfected PTP-PEST from lysates containing approximately 0.375 mg protein.

Substrate trapping was performed using various PTP affinity matrices. The full-length PTP-PEST matrix utilized covalent coupled AG25-protein A-Sepharose beads to which purified baculovirus-expressed PTP-PEST protein was bound. Aliquots (10 ml) of AG25 beads were incubated for 2 hours at 4° C. in 100 ml buffer A in the presence of 5 mg of purified PTP-PEST (wild type or mutant forms); unbound PTP-PEST was then removed by washing three times with 1 ml buffer A. The resultant PTP-PEST-AG25-protein A-Sepharose beads contained approximately 2 mg of PTP-PEST per 10 ml aliquot. Substrate trapping was also carried out with glutathione-Sepharose beads bound to bacterially-expressed GST fusion proteins containing the catalytic domain of PTP-PEST.

PTP1B was also used in substrate trapping experiments. In this case, the monoclonal antibody FG6 was precoupled to protein A-Sepharose in the absence of cross-linker (2 mg antibody/10 ml beads), then purified PTP1B proteins were added in excess and incubated at 4° C. for 2 hours. Following removal of unbound PTP1B, 10 ml beads contained approximately 2 mg PTP1B.

Pervanadate-treated cell lysates, or column fractions, were used as a source of phosphotyrosine-containing proteins for substrate trapping experiments. In general, lysates containing 0.25-0.5 mg protein in 0.5 ml buffer A (including 5 mM iodoacetic acid, 10 mM DTT) were incubated at 4° C. for 2 hours in the presence of 10 ml of affinity matrix containing approximately 2 mg of the appropriate PTP protein. Unbound proteins were then removed from the samples by washing three times with 1 ml buffer A, and bound material was collected by addition of 50 ml SDS-PAGE sample buffer followed by heating at 95° C. for 5 minutes; proteins bound to the beads were then analyzed by SDS-PAGE followed by immunoblotting.

Results

The following details the results of the work described herein carried out as described above.

PTP1B and p210bcr:ab1

Chronic myelogenous leukemia (CML) is a clonal disorder of the haematopoietic stem cell that is characterized by the Philadelphia chromosome, in which the c-Abl proto-oncogene on chromosome 9, encoding a PTK, becomes linked to the bcr gene on chromosome 22. This results in the generation of a bcr:able fusion protein, p210 bcr:abl, in which the PTK activity is enhanced relative to that of c-Abl. Current data indicates that this cytogenetic abnormality is the primary and sole causative event in CML. Expression of p210 bcr:abl produces abnormal patterns of tyro sine phosphorylation that result in the aberrant maturation of the haematopoietic stem cell that is characteristic of CML.

Expression of PTP1B mRNA and protein is enhanced as a consequence of p210 bcr:abl expression in Rat1, Mo7 and BaF3 cells. Changes in PTP1B activity, which were commensurate with the change in enzyme protein, were also observed. These changes are specific for PTP1B and are not seen in closely related homologue (65% identity) TC-PTP or in other tested PTPs, including SHP-1, SHP-2 and PTP-PEST. The increase in expression of PTPIB was also observed in Ph+B-lymphoid cells derived from a CML patient relative to Ph- cells from the same patient.

The changes in PTP1B levels were induced specifically by p210 bcr:abl and were not seen in cells expressing other PTKs including v-abl, v-src or other oncoproteins such as myc. The PTK activity of p210bcr:abl was essential for the increase in expression of PTP1B, since expression of an inactive lysine to arginine mutant form of p210 bcr:abl in Rat1 cells did not alter PTP1B levels. The increase in PTP1B levels is a rapid response to induction of p210 bcr:abl. When BaF3 cells expressing a temperature-sensitive mutant form of p210 bcr:abl were shifted to the permissive temperature for the PTK, PTP1B levels were observed to increase within 12-24 hours coincident with the appearance of the active form of the PTK. These data indicate that the alteration in PTP1B levels is a relatively rapid response to the appearance of p210 bcr:abl, rather than a long-term adaptive response of the cells.

In transient cotransfection experiments in COS cells, PTP1B dephosphorylates p210 bcr:abl but not v-abl. When the PTP1B D181A mutant was expressed as a GST fusion protein, purified and incubated with lysates of Mo7-p210 cells (which overexpress p210 bcr:abl), a complex of the mutant PTP and p210 bcr:abl was isolated. In contrast, tyrosine phosphorylated c-abl, which was also present in the lysates, did not bind to the mutant PTP. The interaction between PTP1B D181A and p210 bcr:abl was blocked by vanadate, suggesting that the interaction involved the active site of the PTP.

Following transient coexpression in COS cells, PTP1B D181A formed a complex with p210 bcr:abl. Preliminary data indicate that the Y177F mutant form of p210 bcr:abl did not interact with PTP1b D181A, suggesting that this tyrosine residue is a component of the binding site in the PTK. This tyrosine residue in p210 bcr:abl is phosphorylated in vivo and has been demonstrated to serve as a docking site for GRB2. Direct interaction of the pTyr in p210 bcr:abl and the SH2 domain of GRB2 is essential for the transforming activity of the PTK. Interaction of PTP1B D181 A with p210 bcr:abl interferes with the association of the PTK with GRB2. Taken together, these data suggest that p210 bcr:abl is a physiological substrate of PTP1B and that PTP1B may function as an antagonist of the oncoprotein PTK in vivo. The Vmax, Km and Kcat of 37 kDa PTP1B mutants toward RCML are shown in FIG. 2.

PTP1B and the EGF Receptor

Expression of PTP1B D181A in COS cells leads to enhanced phosphorylation of tyrosyl residues in a 180 kDa protein and in proteins of 120 and 70 kDa. When a GST-PTP1B D181A fusion protein is expressed in COS cells and precipitated on Glutathione-Sepharose, the 180 kDa, and smaller quantities of p120 nd p70, were coprecipitated. The p180 protein was identified as the epidermal growth factor (EGF) receptor by immunoblotting. The identity of the p120 and p70 proteins is unclear; however, the latter is not src, p62 or paxillin.

Expression of PTP1B D181A in COS cells induces tyrosine phosphorylation of the EGF receptor in the absence of its ligand, EGF, indicating that the mutant PTP is exerting its effects in the intact cell and not post-lysis. The equivalent D199A PTP-PEST mutant does not interact with the EGF receptor, indicating the specificity of this substrate interaction.

Autophosphorylation of the EGF receptor is required for the interaction with PTP1B D181A. Mutants of the receptor that are either kinase-dead or in which the autophosphorylation sites have been deleted do not interact with PTP1B D181A. In v-src-expressing cells, a plethora of tyrosine phosphorylated proteins were observed, but phosphorylation of the EGF receptor was not detected. Under these conditions, PTP1B D181A bound predominantly to a 70 kDa tyrosine phosphorylated protein.

As a result of this work, it appears that PTP 1B can modulate EGF-induced signalling pathways, perhaps including the pathways of many diseases, including breast cancer.

Preferential Dephosphorylation of a 130 kDa Phosphotyrosine-Containing Protein by PTP-PEST

In order to investigate the substrate specificity of PTP-PEST in vitro, aliquots of pervanadate-treated HeLa cell lysates were incubated on ice, yielding 50-100 distinct phosphotyrosine-containing proteins as judged by immunoblotting of the cell lysate using the monoclonal anti-phosphotyrosine antibody G104. Purified full-length PTP-PEST (expressed in Sf9 cells using recombinant baculovirus), PTP-PEST catalytic domain, or PTP1B catalytic domain (37 kDa form) was then added to the lysate, and aliquots were removed at various time points for analysis by SDS-PAGE followed by anti-phosphotyrosine immunoblotting.

Surprisingly, a prominent 130 kDa phosphotyrosine band (p130) was selectively dephosphorylated by PTP-PEST within 10 minutes, whereas the intensity of all the other bands was essentially unchanged even after 60 minutes of incubation with PTP-PEST. Long incubations with higher concentrations of PTP-PEST (greater than 100-fold) resulted in the complete removal of all phosphotyrosine bands from the lysate. However, under all conditions tested, p130 was found to be dephosphorylated more rapidly than all other bands present.

The selective dephosphorylation of p130 by PTP-PEST was also observed using a truncated form of the phosphatase (amino acid residues 1-305) which essentially contains only the catalytic domain of the enzyme. This result suggests that the striking substrate preference displayed by PTP-PEST in this analysis is an inherent property of the phosphatase catalytic domain, whereas the C-terminal 500 amino acid residues have little discernible effect on the substrate specificity of the enzyme.

The specificity of the interaction between PTP-PEST and p130 was addressed using the catalytic domain of PTP1B (amino acid residues 1-321) in dephosphorylation reactions. When added at a similar molar concentration to that used for PTP-PEST, PTP1B was found to dephosphorylate fully and rapidly (within 15 minutes) most of the phosphotyrosine-containing proteins present in the pervanadate-treated HeLa lysate. In addition, the time course of dephosphorylation of p130 was not significantly more rapid than that of the other phosphotyrosine bands dephosphorylated by PTP1B. It should be noted, however, that these in vitro dephosphorylation results are not truly illustrative of the substrate specificity of PTP1B in vivo for several reasons. First, only the isolated catalytic subunit was used in this particular experiment. Furthermore, in vivo substrate specificity may be quite different due to the intracellular distribution of both the PTP and potential substrates. That is, in vitro dephosphorylation experiments may utilize substrates which the PTP is capable of dephosphorylating but which it would not have access to in vivo. The phenomenon of differing substrate specificity depending upon different physiologic contexts is illustrated by a comparison of this data with the in vivo PTP1B work described above, wherein PTP1B showed specificity for only three proteins.

Identification of Phosphotyrosine-Containing p130 Protein as p130^(cas) by Substrate Trapping

Pervanadate-treated HeLa cell lysate was fractionated by anion exchange chromatography and aliquots of the fractions were analyzed by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine or anti-p130^(cas) antibodies. Aliquots of all samples analyzed were then incubated with an affinity matrix containing a substrate trapping PTP-PEST mutant, comprising full length PTP-PEST in which Asp199 is changed to alanine (D199A), bound to covalently coupled protein A-Sepharose/antibody (AG25) beads. Proteins associated with PTP-PEST were then analyzed by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine or anti-p130^(cas) antibodies.

Anti-phosphotyrosine immunoblotting of the column fractions showed that the p130 phosphotyrosine band eluted as a single peak in fractions 11-14 (approx. 0.3 M NaCl). In view of the abundance of tyrosine phosphorylated p130 in HeLa lysates, it appeared likely that p130 represents a previously identified phosphotyrosine-containing 130 kDa protein. Several potential candidates were identified in the literature, including the focal adhesion kinase p125^(FAK), ras-GAP, gp130 and p130^(cas). Of these candidates, p130^(cas) has been identified as a particularly prominent phosphotyrosine band in a wide variety of systems, including v-crk (Mayer and Hanafusa, Proc. Natl. Acad. Sci. USA 87: 2638-2642 (1990); Mayer et al., Nature 332:272-275 (1988)) and src (Kanner et al., Proc. Natl. Acad. Sci. USA 87:3328-3332 (1990); Reynolds et al., Mol. Cell. Biol. 9: 3951-3958 (1989)) transformed fibroblasts, integrin-mediated cell adhesion (Nojima et al., J. Biol. Chem. 270:15398-15402 (1995); Petch et al., J. Cell Science 108:1371-1379 (1995); Vuori and Ruoslahti, J. Biol. Chem. 270:22259-22262 (1995)) and PDGF stimulated 3T3 cells (Rankin and Rozengurt, J. Biol. Chem. 269:704-710 (1994)).

Therefore, the possibility that the p130 phosphotyrosine band corresponds to p130^(cas) was tested by immunoblotting the Mono Q fractions using an antibody to p130^(cas). The 130 kDa band corresponding to p130^(cas) eluted in the same fractions as the p130 tyrosine phosphorylated band, and displayed a similar apparent molecular weight, suggesting that they might represent the same protein. Furthermore, p130^(cas) immunoprecipitated from these fractions was found to be phosphorylated on tyrosyl residues.

A mutant form of PTP-PEST (D199A) was generated by site-directed mutagenesis, and the mutant enzyme was purified following expression using recombinant baculovirus. When assayed using tyrosine phosphorylated RCM-Lysozyme as substrate, the purified mutant enzyme exhibited a specific activity which was approximately 10,000 fold lower than that of the wild type enzyme (Garton and Tonks, unpublished data). This purified protein was bound to an affinity matrix comprised of an anti-PTP-PEST monoclonal antibody (AG25) covalently coupled to Protein A-Sepharose beads, then incubated with each of the Mono Q fractions. After 45 minutes of incubation, proteins associating with the mutant PTP-PEST were collected by centrifugation, the beads were washed, and SDS-PAGE sample buffer was added. Associated proteins were then analyzed by immunoblotting using the monoclonal anti-phosphotyrosine antibody G104.

The mutant PTP-PEST protein was found to associate with a single phosphotyrosine-containing protein, the molecular weight (130 kDa) and Mono Q elution position (fractions 11-14) of which coincided with those of p130^(cas). Immunoblotting of the PTP-PEST-associated proteins using the p130^(cas) antibody demonstrated that the 130 kDa tyrosine phosphorylated protein trapped by the mutant PTP-PEST is indeed p130^(cas). These data further support the hypothesis that p130^(cas) is a potential physiologically relevant substrate for PTP-PEST.

Determination of Structural Features of PTP-PFST Involved in Specific Interaction with Tyrosine Phosphorylated p130^(cas)

The interaction between p130^(cas) and PTP-PEST was investigated further in substrate trapping experiments using various purified mutant forms of PTP-PEST to precipitate proteins from pervanadate-treated HeLa lysates. Several affinity matrices were incubated with pervanadate-treated HeLa cell lysate, and proteins associated with the beads were analyzed by SDS-PAGE followed by immunoblotting with anti-phosphotyrosine or anti-p130^(cas) antibodies.

The wild type full-length phosphatase was found to be incapable of stable association with tyrosine phosphorylated p130^(cas), whereas both the PTP-PEST (D199A) mutant protein and a mutant lacking the active site cysteine residue (C231S) specifically precipitated p130^(cas) from the lysate. The inability of the wild type phosphatase to precipitate tyrosine phosphorylated p130^(cas) presumably reflects the transient nature of the normal interaction between PTP-PEST and tyrosine phosphorylated p130^(cas), which is likely to be concluded as soon as p130^(cas) is dephosphorylated by PTP-PEST.

Since the C-terminal 500 amino acids of PTP-PEST contain several proline-rich regions which resemble src homology-3 (SH3) domain binding sequences, it appeared plausible that the specificity of the interaction between PTP-PEST and p130^(cas) might depend to some extent on association of these segments with the SH3 domain of p130^(cas). The possible contribution of the C-terminal segment of PTP-PEST in the observed specific interaction of PTP-PEST with p130^(cas) was therefore addressed in further substrate trapping experiments using GST fusion proteins containing the catalytic domain of PTP-PEST alone, in both wild type and mutant (D199A) forms. The mutant catalytic domain of PTP-PEST fused to GST was found to precipitate the p130^(cas) phosphotyrosine band specifically, whereas both the wild type fusion protein and GST alone failed to precipitate p130^(cas). The specific interaction between PTP-PEST and p130^(cas) observed in these experiments therefore appears to be an intrinsic property of the catalytic domain of PTP-PEST, emulating the observed preference of the active PTP-PEST catalytic domain for dephosphorylation of p130^(cas) in vitro.

Specificity of Interaction Between Mutant PTP-PEST and Tyrosine Phosphorylated p130^(cas)

In view of the relative abundance of tyrosine phosphorylated p130^(cas) in the pervanadate-treated HeLa cell lysate, the possibility that the observed selective binding of PTP-PEST inactive mutant proteins to p130^(cas) was substrate-directed (reflecting the abundance of this potential substrate relative to the other phosphotyrosine-containing proteins present in the lysate) rather than enzyme-directed (reflecting a genuine substrate preference of PTP-PEST) was considered; this possibility was addressed in two ways. First, inactive mutant forms of the catalytic domain of PTP1B were used to trap potential substrates for this enzyme from the pervanadate-treated HeLa lysates. Again it was found that the wild type phosphatase was incapable of stable interaction with any phosphotyrosine-containing protein, whereas mutant variants of the PTP1B phosphatase domain (comprising Cys or Asp mutations analogous to those described above for PTP-PEST) associated with many tyrosine phosphorylated proteins. This was especially apparent for the aspartic acid mutant of PTP1B (D181A), which appeared to precipitate essentially all phosphotyrosine-containing proteins from the lysate with similar efficacy. These data emphasize the specific nature of the interaction between PTP-PEST and p130^(cas), which appears to be a property peculiar to the PTP-PEST catalytic domain, rather than a feature shared by all PTP catalytic domains.

The specificity of the interaction between PTP-PEST and p130^(cas) was addressed further following pervanadate-treatment of several different cell lines (Wi38, 293, COS, MCF10A, C2C12, MvLu), yielding a different array of tyrosine phosphorylated proteins in each case; the resultant lysates were analyzed by SDS-PAGE followed by anti-phosphotyrosine immunoblotting. Aliquots were incubated with PTP-PEST (D199A) affinity matrix or control matrix, and tyrosine phosphorylated proteins associating with PTP-PEST were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine or anti-p130^(cas) antibodies as described above.

In each case, the D199A mutant PTP-PEST protein precipitated a single broad phosphotyrosine band with an apparent molecular weight between 120 and 150 kDa in different cell lines, whereas the affinity matrix alone failed to precipitate any phosphotyrosine-containing protein. Immunoblotting of the precipitates with a p130^(cas) antibody revealed that the protein precipitated from all cell lysates corresponded to p130^(cas) ; the observed molecular weight variation between different cell lines presumably reflects either species differences in the molecular weight of p130^(cas) or expression of different alternatively spliced forms (Sakai et al., EMBO J. 13:3748-3756 (1994)).

The relative abundance of tyrosine phosphorylated p130^(cas) in the PTP-PEST precipitates appeared to correlate approximately with the abundance of p130^(cas) protein in the lysates (data not shown). Surprisingly, regardless of the abundance of tyrosine phosphorylated p130^(cas) in the lysates, p130^(cas) was invariably the only phosphotyrosine-containing protein in the precipitates, even in 293 cell lysates which contained very little p130^(cas) protein but which displayed a wide variety of other abundantly tyrosine phosphorylated proteins. Similarly, when lysates of pervanadate-treated 293 cells (containing tyrosine phosphorylated p130^(cas) in amounts which are undetectable by anti-phosphotyrosine immunoblotting of the lysate) were incubated with active PTP-PEST, no visible dephosphorylation of any phosphotyrosine band occurred (Garton and Tonks, unpublished data). These results indicate that the affinity of PTP-PEST for p130^(cas) is substantially greater than for any other substrate present, and further emphasizes the remarkable substrate selectivity of PTP-PEST for p 130^(cas).

Vanadate Inhibition of Tyrosine Phosphorylated p130^(cas) Association with Mutant PTP-PEST

A consistent observation of this work was that, in contrast to the inactive mutant PTP-PEST, the wild type enzyme failed to associate in a stable complex with tyrosine phosphorylated p130^(cas), suggesting that the observed association is active site-directed. In order to investigate this possibility, mutant PTP-PEST (D199A) was incubated with the PTP inhibitor vanadate at various concentrations prior to addition of pervanadate-treated HeLa cell lysate. The extent of association of p130^(cas) with PTP-PEST was then analyzed. PTP-PEST affinity matrix, comprising full length PTP-PEST (D199A) bound to covalently coupled protein A-Sepharose/antibody (AG25) beads, was incubated for 10 minutes on ice in the presence of varying concentrations of sodium orthovanadate. The samples were then incubated with aliquots of pervanadate-treated HeLa cell lysate; associated proteins were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine or anti-p130^(cas) antibodies. The activity of wild type PTP-PEST was also determined under the same conditions, using tyrosine phosphorylated ³² P-labelled RCM-lysozyme as substrate.

The association was found to be potently disrupted by vanadate, with a concentration-dependence similar to that of vanadate inhibition of wild type PTP-PEST, and complete disruption being observed at 10 mM vanadate. Since PTP inhibition by vanadate presumably results from a direct interaction of vanadate with the active site cysteine residue of the enzyme (Denu et al., Proc. Natl. Acad. Sci. USA 93:2493-2498 (1996)), this result supports the hypothesis that the stable association of mutant PTP-PEST with tyrosine phosphorylated p130^(cas) is mediated by direct interactions between active site residues within PTP-PEST, in particular the active site cysteine residue, and phosphotyrosine moieties within p130^(cas).

Association of Endogenous p130^(cas) with Transfected Mutant PTP-PEST in COS Cells

The work described above strongly suggests that p130^(cas) represents a potential physiologically significant substrate for PTP-PEST. In order to assess whether PTP-PEST interacts with p130^(cas) in intact cells, COS cells were transfected with plasmids encoding wild type or mutant forms of PTP-PEST (D199A or C215S). The cells were treated with pervanadate 30 minutes prior to lysis, PTP-PEST proteins were immunoprecipitated, and associated tyrosine phosphorylated proteins were analyzed by anti-phosphotyrosine immunoblotting of the resultant precipitates. Lysates were also incubated with covalently coupled protein A-Sepharose/anti-PTP-PEST (AG25) beads and associated proteins were analyzed by SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody.

Under these conditions, the phosphotyrosine-containing band corresponding to p130^(cas) was again unique in its ability to associate with the C231S PTP-PEST protein, indicating that p130^(cas) can be specifically selected by PTP-PEST as a substrate in an intracellular context in the presence of a large number of alternative possible substrates. Neither the wild type nor the D199A form of PTP-PEST was capable of a stable interaction with tyrosine phosphorylated p130^(cas) in pervanadate-treated COS cells.

The binding of both wild type and D199A PTP-PEST to tyrosine phosphorylated p130^(cas) under these conditions is most likely prohibited by the presence of pervanadate bound to the active site cysteine residue of PTP-PEST (Denu et al., Proc. Natl. Acad. Sci. USA 93:2493-2498 (1996)), which effectively excludes the binding of phosphotyrosine residues of p130^(cas). The ability of the C231S mutant PTP-PEST to associate in a stable complex with p130^(cas) in the presence of pervanadate suggests that this mutant protein is largely unaffected by pervanadate, indicating that the normal mode of inhibition of PTPs by vanadate ions depends critically on direct interactions between vanadate and the thiolate anion of the PTP active-site cysteine residue. These observations therefore lend further support to the existence of an exclusive interaction between PTP-PEST and p130^(cas) which appears to be entirely active site-directed, and therefore reflects the genuine, inherent, highly restricted substrate preference of PTP-PEST for p130^(cas).

Results described herein implicate p130^(cas) as a physiologically relevant substrate for PTP-PEST. Furthermore, the observed stringency and exclusivity of the interaction between PTP-PEST and p130^(cas) in a wide variety of cell lines suggest that p130^(cas) may be a unique high affinity substrate for PTP-PEST, although the possibility that other significant PTP-PEST substrates may exist cannot be excluded at present. In particular, it is unclear whether pervanadate-treated cells display a complete spectrum of all possible tyrosine phosphorylated proteins; in fact this appears unlikely since pervanadate treatment presumably results only in an increase in tyrosine phosphorylation of proteins which are to some extent constitutively phosphorylated, but which are normally rapidly dephosphorylated, within the cell. Potential substrates lacking from pervanadate-treated cells therefore presumably include substrates of protein tyrosine kinases (PTKs) which are normally present in an inactive state, such as ligand-stimulated receptor PTKs, and the recently described calcium regulated kinase PYK2 (Lev et al., Nature 376: 737-745 (1995)). Regardless of these considerations, the ability of PTP-PEST to select p130^(cas) exclusively as a substrate from lysates of several different cell lines, containing a combined total of at least one hundred different potential substrates (many of which presumably contain multiple sites of phosphorylation), clearly demonstrates that the substrate specificity of PTP-PEST is highly restricted.

Many intracellular PTPs are limited in their substrate availability due to strict confinement within a particular subcellular location; examples include PTP1B, which is localized to the cytoplasmic face of the endoplasmic reticulum (Frangioni et al., Cell 68:545-560 (1992)), and TCPTP which is either nuclear (Tillmann et al., Mol. Cell. Biol. 14:3030-3040 (1994)) or localized to the endoplasmic reticulum, depending upon which alternative spliced form is expressed (Lorenzen et al., J. Cell Biol. 131:631-643 (1995)). Alternatively, certain PTPs appear to be highly regulated, requiring activation before appreciable activity can be demonstrated. For example, the SH2 domain-containing PTPs, SHP1 and SHP2, display relatively low activity in vitro, but can be considerably activated by several mechanisms including C-terminal truncation (Zhao et al., J. Biol. Chem. 268:2816-2820 (1993)), addition of certain phospholipids (Zhao et al., Proc. Natl. Acad. Sci. USA 90:4251-4255 (1993)), or SH2 domain-mediated binding of appropriate phosphotyrosine-containing peptides (Lechleider et al., J. Biol. Chem. 268:21478-21481 (1993)).

However, PTP-PEST exhibits high specific activity in vitro (35,000 U/mg), and is a predominantly (90-95%) soluble PTP within cells (Garton and Tonks, unpublished data); in principle, therefore, it may act potently on any substrate accessible to the cytoplasm. This accessibility may partly underlie the necessity for PTP-PEST to possess an inherently constrained substrate specificity. The demonstration that mutant PTP-PEST is capable of exclusively associating with p130^(cas) in an intracellular context in the presence of many other tyrosine phosphorylated proteins, is an indication that the narrow substrate specificity of the enzyme may result in PTP-PEST having a negligible influence on the phosphorylation state of the majority of tyrosine phosphorylated proteins within the cell, even though those substrates are largely accessible to PTP-PEST.

The role of p₁₃₀ ^(cas) in cellular transformation by the v-crk and v-src oncogenes is unclear, although there is a general correlation between the level of tyrosine phosphorylation of p130^(cas) and the degree of transformation in cells expressing different forms of crk or src (Kanner et al., EMBO J. 10:1689-1698 (1991); Mayer and Hanafusa, J. Virol. 64:3581-3589 (1990)). Furthermore, enhanced tyrosine phosphorylation of p130cas has also been observed in cells transformed by c-Ha-ras and by ornithine decarboxylase overexpression (Auvinen et al., Mol. Cell. Biol. 15:6513-6525 (1995)). Expression of antisense cDNA encoding p130^(cas) in these cells results in a partial reversion of the transformed phenotype. These observations suggest that aberrant tyrosine phosphorylation of p₁₃₀ ^(cas) is a common feature of cells transformed by several disparate mechanisms and that p130^(cas) may be required for full manifestation of the transformed state. Dephosphorylation of p130^(cas) by PTP-PEST is therefore a potentially important regulatory mechanism for counteracting the transforming effects of various oncogenes.

Tyrosine phosphorylation of p130^(cas) has been observed in fibroblasts following integrin-mediated cell adhesion to extracellular matrix proteins (Nojima et al., J. Biol. Chem. 270:15398-15402 (1995); Petch et al., J. Cell Science 108:1371-1379 (1995); Vuori and Ruoslahti, J. Biol. Chem. 270:22259-22262 (1995)). Under these conditions, using an antibody (4F4) that predominantly recognizes tyrosine phosphorylated p130^(cas) (Kanner et al., EMBO J. 10:1689-1698 (1991); Petch et al., J. Cell Science 108:1371-1379 (1995)) it was shown that phosphorylated p130^(cas) is localized to focal adhesions (Petch et al., J. Cell Science 108:1371-1379 (1995)), whereas fractionation studies have demonstrated that the normal cellular location of the majority of non-phosphorylated p130^(cas) is the cytosol (Sakai et al., EMBO J. 13:3748-3756 (1994)). Furthermore, in crk-transformed fibroblasts, tyrosine phosphorylated p130^(cas) is detected only in insoluble fractions (Sakai et al., EMBO J. 13:3748-3756 (1994)), suggesting that both cell adhesion- and transformation-mediated phosphorylation of p130^(cas) is associated with redistribution of the protein from the cytosol to focal adhesions.

It is plausible that the redistribution of tyrosine phosphorylated p 130^(cas) may be driven by its association with FAK, which is constitutively associated with focal adhesions due to its C-terminal focal adhesion targeting domain (Hildebrand et al., J. Cell Biol. 123:993-1005 (1993); Schaller et al., Proc. Natl. Acad. Sci. USA 89:5192-5196 (1992)). The sequestration of tyrosine phosphorylated p130^(cas) in focal adhesions both in transformed cells, and following integrin-mediated cell adhesion, strongly suggests a role for p130^(cas) in signalling events in this region of the cell. One consequence of the redistribution of tyrosine phosphorylated p130^(cas) is likely to be that, in addition to localizing p130^(cas) to a region of the cell containing abundant protein tyrosine kinase activity, the phosphorylated protein will be relatively inaccessible to the cytosolic phosphatase PTP-PEST. This raises the possibility that the role of PTP-PEST in dephosphorylating p130^(cas) may be to prevent inappropriate tyrosine phosphorylation of the cytosolic pool of p130^(cas), thus preventing formation of signalling complexes assembled around tyrosine phosphorylated p130^(cas) in inappropriate cellular locations.

Several mitogenic factors potently stimulate tyrosine phosphorylation of p130^(cas). These include agents acting through heterotrimeric G protein-coupled receptors such as lysophosphatidic acid (Seufferlein and Rozengurt, J. Biol. Chem. 269:9345-9351 (1994)), bradykinin (Leeb-Lundberg et al., J. Biol. Chem. 269: 24328-24344 (1994)), and bombesin (Zachary et al., J. Biol. Chem. 267:19031-19034 (1992)), as well as growth factors that activate receptor tyrosine kinases, namely PDGF (Rankin and Rozengurt, J. Biol. Chem. 269:704-710 (1994)), EGF and NGF (Ribon and Saltiel, J. Biol. Chem. 271:7375-7380 (1996)). These observations suggest roles for p130^(cas) in regulation of mitogenic signalling pathways, presumably involving assembly of signalling complexes based on tyrosine phosphorylated p130^(cas). The identities of the proteins involved in these complexes are not established, but are likely to include SH2 domain-containing adaptor proteins such as crk (Ribon and Saltiel, J. Biol. Chem. 271:7375-7380 (1996)), and its associated proteins (Feller et al., Oncogene 10:1465-1473 (1995); Hasegawa et al., Mol. Cell. Biol. 16:1770-1776 (1996); Knudsen et al., J. Biol. Chem. 269:32781-32787 (1994); Matsuda et al., Mol. Cell. Biol. 14: 5495-5500 (1994); Tanaka et al., Proc. Natl. Acad. Sci. USA 91:3443-3447 (1994)). Therefore tyrosine phosphorylation and dephosphorylation of p130^(cas) potentially plays a central role in regulating the formation of such complexes, thereby influencing downstream events in mitogenic signalling.

Equivalents

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

    __________________________________________________________________________     #             SEQUENCE LISTING     - (1) GENERAL INFORMATION:     -    (iii) NUMBER OF SEQUENCES: 36     - (2) INFORMATION FOR SEQ ID NO:1:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 196 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:     - Asp Phe Pro Cys Arg Val Ala Lys Leu Pro Ly - #s Asn Lys Asn Arg Asn     #                15     - Arg Tyr Arg Asp Val Ser Pro Phe Asp His Se - #r Arg Ile Lys Leu His     #            30     - Gln Glu Asp Asn Asp Tyr Ile Asn Ala Ser Le - #u Ile Lys Met Glu Glu     #        45     - Ala Gln Arg Ser Tyr Ile Leu Thr Gln Gly Pr - #o Leu Pro Asn Thr Cys     #    60     - Gly His Phe Trp Glu Met Val Trp Glu Gln Ly - #s Ser Arg Gly Val Val     #80     - Met Leu Asn Arg Val Met Glu Lys Gly Ser Le - #u Lys Cys Ala Gln Tyr     #                95     - Trp Pro Gln Lys Glu Glu Lys Glu Met Ile Ph - #e Glu Asp Thr Asn Leu     #           110     - Lys Leu Thr Leu Ile Ser Glu Asp Ile Lys Se - #r Tyr Tyr Thr Val Leu     #       125     - Glu Leu Glu Asn Leu Thr Thr Gln Glu Thr Ar - #g Glu Ile Leu His Phe     #   140     - His Tyr Thr Thr Trp Pro Asp Phe Gly Val Pr - #o Glu Ser Pro Ala Ser     145                 1 - #50                 1 - #55                 1 -     #60     - Phe Leu Asn Phe Leu Phe Lys Val Arg Glu Se - #r Gly Ser Leu Ser Pro     #               175     - Glu His Gly Pro Val Val Val His Cys Ser Al - #a Gly Ile Gly Arg Ser     #           190     - Gly Thr Phe Cys             195     - (2) INFORMATION FOR SEQ ID NO:2:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 194 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:     - Asp Tyr Pro His Arg Val Ala Lys Phe Pro Gl - #u Asn Arg Asn Arg Asn     #                15     - Arg Tyr Arg Asp Val Ser Pro Tyr Asp His Se - #r Arg Val Leu Gln Asn     #            30     - Ala Glu Asn Asp Tyr Ile Asn Ala Ser Leu Va - #l Asp Ile Glu Glu Ala     #        45     - Gln Arg Ser Tyr Ile Leu Thr Gln Gly Pro Le - #u Pro Asn Thr Cys Cys     #    60     - His Phe Trp Leu Met Val Trp Gln Gln Lys Th - #r Lys Ala Val Val Met     #80     - Leu Asn Arg Ile Val Glu Lys Glu Ser Val Ly - #s Cys Ala Gln Tyr Trp     #                95     - Pro Thr Asp Asp Gln Glu Met Leu Phe Lys Gl - #u Thr Gly Phe Ser Val     #           110     - Lys Leu Leu Ser Glu Asp Val Lys Ser Tyr Ty - #r Thr Val Leu Gln Leu     #       125     - Glu Asn Ile Asn Ser Gly Glu Thr Arg Thr Il - #e Ser His Phe His Tyr     #   140     - Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Se - #r Pro Ala Ser Phe Leu     145                 1 - #50                 1 - #55                 1 -     #60     - Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Se - #r Leu Asn Pro Asp His     #               175     - Gly Pro Ala Val Ile His Cys Ser Ala Gly Il - #e Gly Arg Ser Gly Thr     #           190     - Phe Ser     - (2) INFORMATION FOR SEQ ID NO:3:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 261 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:     - Gly Ile Thr Ala Asp Ser Ser Asn His Pro As - #p Asn Lys His Lys Asn     #                15     - Arg Tyr Ile Asn Ile Val Ala Tyr Asp His Se - #r Arg Val Lys Leu Ala     #            30     - Gln Leu Ala Glu Lys Asp Gly Lys Leu Thr As - #p Tyr Ile Asn Ala Asn     #        45     - Tyr Val Asp Gly Tyr Asn Arg Pro Lys Ala Ty - #r Ile Ala Ala Gln Gly     #    60     - Pro Leu Lys Ser Thr Ala Glu Asp Phe Trp Ar - #g Met Ile Trp Glu His     #80     - Asn Val Glu Val Ile Val Met Ile Thr Asn Le - #u Val Glu Lys Gly Arg     #                95     - Arg Lys Cys Asp Gln Tyr Trp Pro Pro Ala As - #p Gly Ser Glu Glu Tyr     #           110     - Gly Asn Phe Leu Val Thr Gln Lys Ser Val Gl - #n Val Leu Ala Tyr Tyr     #       125     - Thr Val Phe Thr Leu Arg Asn Thr Lys Ile Ly - #s Lys Gly Ser Gln Lys     #   140     - Gly Arg Pro Ser Gly Arg Val Val Thr Gln Ty - #r His Tyr Thr Gln Trp     145                 1 - #50                 1 - #55                 1 -     #60     - Pro Asp Met Gly Val Pro Glu Tyr Ser Leu Pr - #o Val Leu Thr Phe Val     #               175     - Arg Lys Ala Ala Tyr Ala Lys Arg His Ala Va - #l Gly Pro Val Val Val     #           190     - His Cys Ser Ala Gly Val Gly Arg Thr Gly Th - #r Tyr Ile Val Leu Asp     #       205     - Ser Met Leu Gln Gln Ile Gln His Glu Gly Th - #r Val Asn Ile Phe Gly     #   220     - Phe Leu Lys His Ile Arg Ser Gln Arg Asn Ty - #r Leu Val Gln Thr Glu     225                 2 - #30                 2 - #35                 2 -     #40     - Glu Gln Tyr Val Phe Ile His Asp Thr Leu Va - #l Glu Ala Ile Leu Ser     #               255     - Lys Glu Thr Glu Val                 260     - (2) INFORMATION FOR SEQ ID NO:4:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 260 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:     - Gly Ile Thr Ala Asp Ser Ser Asn His Pro As - #p Asn Lys His Lys Asn     #                15     - Arg Tyr Ile Asn Ile Val Ala Tyr Asp His Se - #r Arg Val Lys Leu Ala     #            30     - Gln Leu Ala Glu Lys Asp Gly Lys Leu Thr As - #p Tyr Ile Asn Ala Asn     #        45     - Tyr Val Asp Gly Tyr Asn Arg Pro Lys Ala Ty - #r Ile Ala Ala Gln Gly     #    60     - Pro Leu Lys Ser Thr Ala Glu Asp Phe Trp Ar - #g Met Ile Trp Glu His     #80     - Asn Val Glu Val Ile Val Met Ile Thr Asn Le - #u Val Glu Lys Gly Arg     #                95     - Arg Lys Cys Asp Gln Tyr Trp Pro Ala Asp Gl - #y Ser Glu Glu Tyr Gly     #           110     - Asn Phe Leu Val Thr Gln Lys Ser Val Gln Va - #l Leu Ala Tyr Tyr Thr     #       125     - Val Phe Thr Leu Arg Asn Thr Lys Ile Lys Ly - #s Gly Ser Gln Lys Gly     #   140     - Arg Pro Ser Gly Arg Val Val Thr Gln Tyr Hi - #s Tyr Thr Gln Trp Pro     145                 1 - #50                 1 - #55                 1 -     #60     - Asp Met Gly Val Pro Glu Tyr Ser Leu Pro Va - #l Leu Thr Phe Val Arg     #               175     - Lys Ala Ala Tyr Ala Lys Arg His Ala Val Gl - #y Pro Val Val Val His     #           190     - Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Ty - #r Ile Val Leu Asp Ser     #       205     - Met Leu Gln Gln Ile Gln His Glu Gly Thr Va - #l Asn Ile Phe Gly Phe     #   220     - Leu Lys His Ile Arg Ser Gln Arg Asn Tyr Le - #u Val Gln Thr Glu Glu     225                 2 - #30                 2 - #35                 2 -     #40     - Gln Tyr Val Phe Ile His Asp Thr Leu Val Gl - #u Ala Ile Leu Ser Lys     #               255     - Glu Thr Glu Val                 260     - (2) INFORMATION FOR SEQ ID NO:5:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 263 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:     - Asn Ile Thr Ala Glu His Ser Asn His Pro Gl - #u Asn Lys His Lys Asn     #                15     - Arg Tyr Ile Asn Ile Leu Ala Tyr Asp His Se - #r Arg Val Lys Leu Arg     #            30     - Pro Leu Pro Gly Lys Asp Ser Lys His Ser As - #p Tyr Ile Asn Ala Asn     #        45     - Tyr Val Asp Gly Tyr Asn Lys Ala Lys Ala Ty - #r Ile Ala Thr Gln Gly     #    60     - Pro Leu Lys Ser Thr Phe Glu Asp Phe Trp Ar - #g Met Ile Trp Glu Gln     #80     - Asn Thr Gly Ile Ile Val Met Ile Thr Asn Le - #u Val Glu Lys Gly Arg     #                95     - Arg Lys Cys Asp Gln Tyr Trp Pro Thr Glu As - #n Ser Glu Glu Tyr Gly     #           110     - Asn Ile Ile Val Thr Leu Lys Ser Thr Lys Il - #e His Ala Cys Tyr Thr     #       125     - Val Phe Ser Ile Arg Asn Thr Lys Val Lys Ly - #s Gly Gln Lys Gly Asn     #   140     - Pro Lys Gly Arg Gln Asn Glu Arg Val Val Il - #e Gln Tyr His Tyr Thr     145                 1 - #50                 1 - #55                 1 -     #60     - Gln Trp Pro Asp Met Gly Val Pro Glu Tyr Al - #a Leu Pro Val Leu Thr     #               175     - Phe Val Arg Arg Ser Ser Ala Ala Arg Met Pr - #o Glu Thr Gly Pro Val     #           190     - Leu Val His Cys Ser Ala Gly Val Gly Arg Th - #r Gly Thr Tyr Ile Val     #       205     - Ile Asp Ser Met Leu Gln Gln Ile Lys Asp Ly - #s Ser Thr Val Asn Val     #   220     - Leu Gly Phe Leu Lys His Ile Arg Thr Gln Ar - #g Asn Tyr Leu Val Gln     225                 2 - #30                 2 - #35                 2 -     #40     - Thr Glu Glu Gln Tyr Ile Phe Ile His Asp Al - #a Leu Leu Glu Ala Ile     #               255     - Leu Gly Lys Glu Thr Glu Val                 260     - (2) INFORMATION FOR SEQ ID NO:6:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 257 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:     - Asp Leu Pro Cys Glu His Ser Gln His Pro Gl - #u Asn Lys Arg Lys Asn     #                15     - Arg Tyr Leu Asn Ile Thr Ala Tyr Asp His Se - #r Arg Val His Leu His     #            30     - Pro Thr Pro Gly Gln Lys Lys Asn Leu Asp Ty - #r Ile Asn Ala Asn Phe     #        45     - Ile Asp Gly Tyr Gln Lys Gly His Ala Phe Il - #e Gly Thr Gln Gly Pro     #    60     - Leu Pro Asp Thr Phe Asp Cys Phe Trp Arg Me - #t Ile Trp Glu Gln Arg     #80     - Val Ala Ile Ile Val Met Ile Thr Asn Leu Va - #l Glu Arg Gly Arg Arg     #                95     - Lys Cys Asp Met Tyr Trp Pro Lys Asp Gly Va - #l Glu Thr Tyr Gly Val     #           110     - Ile Gln Val Lys Leu Ile Glu Glu Glu Val Me - #t Ser Thr Tyr Thr Val     #       125     - Leu Gln Ile Lys His Leu Lys Leu Lys Lys Ly - #s Lys Gln Cys Asn Thr     #   140     - Glu Lys Leu Val Tyr Gln Tyr His Tyr Thr As - #n Trp Pro Asp His Gly     145                 1 - #50                 1 - #55                 1 -     #60     - Thr Pro Asp His Pro Leu Pro Val Leu Asn Ph - #e Val Lys Lys Ser Ser     #               175     - Ala Ala Asn Pro Ala Glu Ala Gly Pro Ile Va - #l Val His Cys Ser Ala     #           190     - Gly Val Gly Arg Thr Gly Thr Tyr Ile Val Le - #u Asp Ala Met Leu Lys     #       205     - Gln Ile Gln Gln Lys Asn Ile Val Asn Val Ph - #e Gly Phe Leu Arg His     #   220     - Ile Arg Ala Gln Arg Asn Phe Leu Val Gln Th - #r Glu Glu Gln Tyr Ile     225                 2 - #30                 2 - #35                 2 -     #40     - Phe Leu His Asp Ala Leu Val Glu Ala Ile Al - #a Ser Gly Glu Thr Asn     #               255     - Leu     - (2) INFORMATION FOR SEQ ID NO:7:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 250 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:     - Gln Phe Thr Trp Glu Asn Ser Asn Leu Glu Va - #l Asn Lys Pro Lys Asn     #                15     - Arg Tyr Ala Asn Val Ile Ala Tyr Asp His Se - #r Arg Val Ile Leu Thr     #            30     - Ser Ile Asp Gly Val Pro Gly Ser Asp Tyr Il - #e Asn Ala Asn Tyr Ile     #        45     - Asp Gly Tyr Arg Lys Gln Asn Ala Tyr Ile Al - #a Thr Gln Gly Pro Leu     #    60     - Pro Glu Thr Met Gly Asp Phe Trp Arg Met Va - #l Trp Glu Gln Arg Thr     #80     - Ala Thr Val Val Met Met Thr Arg Leu Glu Gl - #u Lys Ser Arg Val Lys     #                95     - Cys Asp Gln Tyr Trp Pro Ala Arg Gly Thr Gl - #u Thr Cys Gly Leu Ile     #           110     - Gln Val Thr Leu Leu Asp Thr Val Glu Leu Al - #a Thr Tyr Thr Val Phe     #       125     - Ala Leu His Lys Ser Gly Ser Ser Glu Lys Ar - #g Glu Leu Arg Gln Phe     #   140     - Gln Phe Met Ala Trp Pro Asp His Gly Val Pr - #o Glu Tyr Pro Thr Pro     145                 1 - #50                 1 - #55                 1 -     #60     - Ile Leu Ala Phe Leu Arg Arg Val Lys Ala Cy - #s Asn Pro Leu Asp Ala     #               175     - Gly Pro Met Val Val His Cys Ser Ala Gly Va - #l Gly Arg Thr Gly Cys     #           190     - Phe Ile Val Ile Asp Ala Met Leu Glu Arg Me - #t Lys His Glu Lys Thr     #       205     - Val Asp Ile Tyr Gly His Val Thr Cys Met Ar - #g Ser Gln Arg Asn Tyr     #   220     - Met Val Gln Thr Glu Asp Gln Tyr Val Phe Il - #e His Glu Ala Leu Leu     225                 2 - #30                 2 - #35                 2 -     #40     - Glu Ala Ala Thr Cys Gly His Thr Glu Val     #               250     - (2) INFORMATION FOR SEQ ID NO:8:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 249 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:     - Ser Ala Pro Trp Asp Ser Ala Lys Lys Asp Gl - #u Asn Arg Met Lys Asn     #                15     - Arg Tyr Gly Asn Ile Ile Ala Tyr Asp His Se - #r Arg Val Arg Leu Gln     #            30     - Thr Ile Glu Gly Asp Thr Asn Ser Asp Tyr Il - #e Asn Gly Asn Tyr Ile     #        45     - Asp Gly Tyr His Arg Pro Asn His Tyr Ile Al - #a Thr Gln Gly Pro Met     #    60     - Gln Glu Thr Ile Tyr Asp Phe Trp Arg Met Va - #l Trp His Glu Asn Thr     #80     - Ala Ser Ile Ile Met Val Thr Asn Leu Val Gl - #u Val Gly Arg Val Lys     #                95     - Cys Cys Lys Tyr Trp Pro Asp Asp Thr Glu Il - #e Tyr Lys Asp Ile Lys     #           110     - Val Thr Leu Ile Glu Thr Glu Leu Leu Ala Gl - #u Tyr Val Ile Phe Ala     #       125     - Val Glu Lys Arg Gly Val His Glu Ile Arg Gl - #u Ile Arg Gln Phe His     #   140     - Phe Thr Gly Trp Pro Asp His Gly Val Pro Ty - #r His Ala Thr Gly Leu     145                 1 - #50                 1 - #55                 1 -     #60     - Leu Gly Phe Val Arg Gln Val Lys Ser Lys Se - #r Pro Pro Ser Ala Gly     #               175     - Pro Leu Val Val His Cys Ser Ala Gly Ala Gl - #y Arg Thr Gly Cys Phe     #           190     - Ile Val Ile Asp Ile Met Leu Asp Met Ala Gl - #u Arg Glu Gly Val Val     #       205     - Asp Ile Tyr Asn Cys Val Arg Glu Leu Arg Se - #r Arg Arg Val Asn Met     #   220     - Val Gln Thr Glu Glu Gln Tyr Val Phe Ile Hi - #s Asp Ala Ile Leu Glu     225                 2 - #30                 2 - #35                 2 -     #40     - Ala Cys Leu Cys Gly Asp Thr Ser Val                     245     - (2) INFORMATION FOR SEQ ID NO:9:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 254 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:     - Gln Ala Thr Cys Glu Ala Ala Ser Lys Glu Gl - #u Asn Lys Glu Lys Asn     #                15     - Arg Tyr Val Asn Ile Leu Pro Tyr Asp His Se - #r Arg Val His Leu Thr     #            30     - Pro Val Glu Gly Val Pro Asp Ser Asp Tyr Il - #e Asn Ala Ser Phe Ile     #        45     - Asn Gly Tyr Gln Glu Lys Asn Lys Phe Ile Al - #a Ala Gln Gly Pro Lys     #    60     - Glu Glu Thr Val Asn Asp Phe Trp Arg Met Il - #e Trp Glu Gln Asn Thr     #80     - Ala Thr Ile Val Met Val Thr Asn Leu Lys Gl - #u Arg Lys Glu Cys Lys     #                95     - Cys Ala Gln Tyr Trp Pro Asp Gln Gly Cys Tr - #p Thr Tyr Gly Asn Ile     #           110     - Arg Val Ser Val Glu Asp Val Thr Val Leu Va - #l Asp Tyr Thr Val Phe     #       125     - Cys Ile Gln Gln Val Gly Asp Met Thr Asn Ar - #g Lys Pro Gln Arg Leu     #   140     - Ile Thr Gln Phe His Phe Thr Ser Trp Pro As - #p Phe Gly Val Pro Phe     145                 1 - #50                 1 - #55                 1 -     #60     - Thr Pro Ile Gly Met Leu Lys Phe Leu Lys Ly - #s Val Lys Ala Cys Asn     #               175     - Pro Gln Tyr Ala Gly Ala Ile Val Val His Cy - #s Ser Ala Gly Val Gly     #           190     - Arg Thr Gly Thr Phe Val Val Ile Asp Ala Me - #t Leu Asp Met Met His     #       205     - Thr Glu Arg Lys Val Asp Val Tyr Gly Phe Va - #l Ser Arg Ile Arg Ala     #   220     - Gln Arg Cys Gln Met Val Gln Thr Asp Met Gl - #n Tyr Val Phe Ile Tyr     225                 2 - #30                 2 - #35                 2 -     #40     - Gln Ala Leu Leu Glu His Tyr Leu Tyr Gly As - #p Thr Glu Leu     #               250     - (2) INFORMATION FOR SEQ ID NO:10:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 253 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:     - Gln Gly Thr Phe Glu Leu Ala Asn Lys Glu Gl - #u Asn Arg Glu Lys Asn     #                15     - Arg Tyr Pro Asn Ile Leu Pro Asn Asp His Se - #r Arg Val Ile Leu Ser     #            30     - Gln Leu Asp Gly Ile Pro Cys Ser Asp Tyr Il - #e Asn Ala Ser Tyr Ile     #        45     - Asp Gly Tyr Lys Glu Lys Asn Lys Phe Ile Al - #a Ala Gln Gly Pro Lys     #    60     - Gln Glu Thr Val Asn Asp Phe Trp Arg Met Va - #l Trp Glu Gln Lys Ser     #80     - Ala Thr Ile Val Met Leu Thr Asn Leu Lys Gl - #u Arg Lys Glu Glu Lys     #                95     - Cys His Gln Tyr Trp Pro Asp Gln Gly Cys Tr - #p Thr Tyr Gly Asn Ile     #           110     - Arg Val Cys Val Glu Asp Cys Val Val Leu Va - #l Asp Tyr Thr Ile Phe     #       125     - Cys Ile Gln Pro Gln Leu Pro Asp Gly Cys Ly - #s Ala Pro Arg Leu Val     #   140     - Ser Gln Leu His Phe Thr Ser Trp Pro Asp Ph - #e Gly Val Pro Phe Thr     145                 1 - #50                 1 - #55                 1 -     #60     - Pro Ile Gly Met Leu Lys Phe Leu Lys Lys Va - #l Lys Thr Leu Asn Pro     #               175     - Val His Ala Gly Pro Ile Val Val His Cys Se - #r Ala Gly Val Gly Arg     #           190     - Thr Gly Thr Phe Ile Val Ile Asp Ala Met Me - #t Ala Met Met His Ala     #       205     - Glu Gln Lys Val Asp Val Phe Glu Phe Val Se - #r Arg Ile Arg Asn Gln     #   220     - Arg Pro Gln Met Val Gln Thr Asp Met Gln Ty - #r Thr Phe Ile Tyr Gln     225                 2 - #30                 2 - #35                 2 -     #40     - Ala Leu Leu Glu Tyr Tyr Leu Tyr Gly Asp Th - #r Glu Leu     #               250     - (2) INFORMATION FOR SEQ ID NO:11:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 253 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:     - Lys Phe Pro Ile Lys Asp Ala Arg Lys Pro Hi - #s Asn Gln Asn Lys Asn     #                15     - Arg Tyr Val Asp Ile Leu Pro Tyr Asp Tyr As - #n Arg Val Glu Leu Ser     #            30     - Glu Ile Asn Gly Asp Ala Gly Ser Thr Tyr Il - #e Asn Ala Ser Tyr Ile     #        45     - Asp Gly Phe Lys Glu Pro Arg Lys Tyr Ile Al - #a Ala Gln Gly Pro Arg     #    60     - Asp Glu Thr Val Asp Asp Phe Trp Arg Met Il - #e Trp Glu Gln Lys Ala     #80     - Thr Val Ile Val Met Val Thr Arg Cys Glu Gl - #u Gly Asn Arg Asn Lys     #                95     - Cys Ala Glu Tyr Trp Pro Ser Met Glu Glu Gl - #y Thr Arg Ala Phe Lys     #           110     - Asp Ile Val Val Thr Ile Asn Asp His Lys Ar - #g Cys Pro Asp Tyr Ile     #       125     - Ile Leu Asn Val Ala His Lys Lys Glu Lys Al - #a Thr Gly Arg Glu Val     #   140     - Thr His Ile Gln Phe Thr Ser Trp Pro Asp Hi - #s Gly Val Pro Glu Asp     145                 1 - #50                 1 - #55                 1 -     #60     - Pro His Leu Leu Leu Lys Leu Arg Arg Arg Va - #l Asn Ala Phe Ser Asn     #               175     - Phe Phe Ser Gly Pro Ile Val Val His Cys Se - #r Ala Gly Val Gly Arg     #           190     - Thr Gly Thr Tyr Ile Gly Ile Asp Ala Met Le - #u Glu Gly Leu Glu Ala     #       205     - Glu Gly Lys Val Asp Val Tyr Gly Tyr Val Va - #l Lys Leu Arg Arg Gln     #   220     - Arg Cys Leu Met Val Gln Val Glu Ala Gln Ty - #r Ile Leu Ile His Gln     225                 2 - #30                 2 - #35                 2 -     #40     - Ala Leu Val Glu Tyr Asn Gln Phe Gly Glu Th - #r Glu Val     #               250     - (2) INFORMATION FOR SEQ ID NO:12:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 266 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:     - Leu Tyr Ser Arg Lys Glu Gly Gln Arg Gln Gl - #u Asn Lys Asn Lys Asn     #                15     - Arg Tyr Lys Asn Ile Leu Pro Phe Asp His Th - #r Arg Val Val Leu His     #            30     - Asp Gly Asp Pro Asn Glu Pro Val Ser Asp Ty - #r Ile Asn Ala Asn Ile     #        45     - Ile Met Pro Glu Phe Glu Thr Lys Cys Asn As - #n Ser Lys Pro Lys Lys     #    60     - Ser Tyr Ile Ala Thr Gln Gly Cys Leu Gln As - #n Thr Val Asn Asp Phe     #80     - Trp Arg Met Val Phe Gln Glu Asn Ser Arg Va - #l Ile Val Met Thr Thr     #                95     - Lys Glu Val Glu Arg Gly Lys Ser Lys Cys Va - #l Lys Tyr Trp Pro Asp     #           110     - Glu Tyr Ala Leu Lys Glu Tyr Gly Val Met Ar - #g Val Arg Asn Val Lys     #       125     - Glu Ser Ala Ala His Asp Tyr Thr Leu Leu Ly - #s Leu Ser Lys Val Gly     #   140     - Gln Gly Asn Thr Glu Arg Thr Val Trp Gln Ty - #r His Phe Arg Thr Trp     145                 1 - #50                 1 - #55                 1 -     #60     - Pro Asp His Gly Val Pro Ser Asp Pro Gly Gl - #y Val Leu Asp Phe Leu     #               175     - Glu Glu Val His His Lys Gln Glu Ser Ile Me - #t Asp Ala Gly Pro Val     #           190     - Val Val His Cys Ser Ala Gly Ile Gly Arg Th - #r Gly Thr Phe Ile Val     #       205     - Ile Asp Ile Leu Ile Asp Ile Ile Arg Glu Ly - #s Gly Val Asp Cys Asp     #   220     - Ile Asp Val Pro Lys Thr Ile Gln Met Val Ar - #g Ser Gln Arg Ser Gly     225                 2 - #30                 2 - #35                 2 -     #40     - Met Val Gln Thr Glu Ala Gln Tyr Arg Phe Il - #e Tyr Met Ala Val Gln     #               255     - His Tyr Ile Glu Thr Leu Gln Arg Arg Ile     #           265     - (2) INFORMATION FOR SEQ ID NO:13:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 263 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:     - Leu His Gln Arg Leu Glu Gly Gln Arg Pro Gl - #u Asn Lys Gly Lys Asn     #                15     - Arg Tyr Lys Asn Ile Leu Pro Phe Asp His Se - #r Arg Val Ile Leu Gln     #            30     - Gly Arg Asp Ser Asn Ile Pro Gly Ser Asp Ty - #r Ile Asn Ala Asn Tyr     #        45     - Ile Lys Asn Gln Leu Leu Gly Pro Asp Glu As - #n Ala Lys Thr Tyr Ile     #    60     - Ala Ser Gln Gly Cys Leu Glu Ala Thr Val As - #n Asp Phe Trp Gln Met     #80     - Ala Trp Gln Glu Asn Ser Arg Val Ile Val Me - #t Thr Thr Arg Glu Val     #                95     - Glu Lys Gly Arg Asn Lys Cys Val Pro Tyr Tr - #p Pro Glu Val Gly Met     #           110     - Gln Arg Ala Tyr Gly Pro Tyr Ser Val Thr As - #n Cys Gly Glu His Asp     #       125     - Thr Thr Glu Tyr Lys Leu Leu Gln Val Ser Pr - #o Leu Asp Asn Gly Asp     #   140     - Leu Ile Arg Glu Ile Trp His Tyr Gln Tyr Le - #u Ser Trp Pro Asp His     145                 1 - #50                 1 - #55                 1 -     #60     - Gly Val Pro Ser Glu Pro Gly Gly Val Leu Se - #r Phe Leu Asp Gln Ile     #               175     - Asn Gln Arg Gln Glu Ser Leu Pro His Ala Gl - #y Pro Ile Ile Val His     #           190     - Cys Ser Ala Gly Ile Gly Arg Thr Gly Thr Il - #e Ile Val Ile Asp Met     #       205     - Leu Met Glu Asn Ile Ser Thr Lys Gly Leu As - #p Cys Asp Ile Asp Ile     #   220     - Gln Lys Thr Ile Gln Met Val Arg Ala Gln Ar - #g Ser Gly Met Val Gln     225                 2 - #30                 2 - #35                 2 -     #40     - Thr Glu Ala Gln Tyr Lys Phe Ile Tyr Val Al - #a Ile Ala Gln Phe Ile     #               255     - Glu Thr Thr Lys Lys Lys Leu                 260     - (2) INFORMATION FOR SEQ ID NO:14:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 254 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:     - Asn Gln Ser Cys Asp Ile Ala Leu Leu Pro Gl - #u Asn Arg Gly Lys Asn     #                15     - Arg Tyr Asn Asn Ile Leu Pro Tyr Asp Ala Th - #r Arg Val Lys Leu Ser     #            30     - Asn Val Asp Asp Asp Pro Cys Ser Asp Tyr Il - #e Asn Ala Ser Tyr Ile     #        45     - Pro Gly Asn Asn Phe Arg Arg Glu Tyr Ile Va - #l Thr Gln Gly Pro Leu     #    60     - Pro Gly Thr Lys Asp Asp Phe Trp Lys Met Va - #l Trp Glu Gln Asn Val     #80     - His Asn Ile Val Met Val Thr Gln Cys Val Gl - #u Lys Gly Arg Val Lys     #                95     - Cys Asp His Tyr Trp Pro Ala Asp Gln Asp Se - #r Leu Tyr Tyr Gly Asp     #           110     - Leu Ile Leu Gln Met Leu Ser Glu Ser Val Le - #u Pro Glu Trp Thr Ile     #       125     - Phe Lys Ile Cys Gly Glu Glu Gln Leu Asp Al - #a His Arg Leu Ile Arg     #   140     - His Phe His Tyr Thr Val Trp Pro Asp His Gl - #y Val Pro Glu Thr Thr     145                 1 - #50                 1 - #55                 1 -     #60     - Gln Ser Leu Ile Gln Phe Val Arg Thr Val Ar - #g Asp Tyr Ile Asn Arg     #               175     - Ser Pro Gly Ala Gly Pro Thr Val Val His Cy - #s Ser Ala Gly Val Gly     #           190     - Arg Thr Gly Thr Phe Ile Ala Leu Asp Arg Il - #e Leu Gln Gln Leu Asp     #       205     - Ser Lys Asp Ser Val Asp Ile Tyr Gly Ala Va - #l His Asp Leu Arg Leu     #   220     - His Arg Val His Met Val Gln Thr Glu Cys Gl - #n Tyr Val Tyr Leu His     225                 2 - #30                 2 - #35                 2 -     #40     - Gln Cys Val Arg Asp Val Leu Arg Ala Arg Ly - #s Leu Arg Ser     #               250     - (2) INFORMATION FOR SEQ ID NO:15:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 251 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:     - Asp Gln Pro Cys Thr Phe Ala Asp Leu Pro Cy - #s Asn Arg Pro Lys Asn     #                15     - Arg Phe Thr Asn Ile Leu Pro Tyr Asp His Se - #r Arg Phe Lys Leu Gln     #            30     - Pro Val Asp Asp Asp Glu Gly Ser Asp Tyr Il - #e Asn Ala Asn Tyr Val     #        45     - Pro Gly His Asn Ser Pro Arg Glu Phe Ile Va - #l Thr Gln Gly Pro Leu     #    60     - His Ser Thr Arg Asp Asp Phe Trp Arg Met Cy - #s Trp Glu Ser Asn Ser     #80     - Arg Ala Ile Val Met Leu Thr Arg Cys Phe Gl - #u Lys Gly Arg Glu Lys     #                95     - Cys Asp Gln Tyr Trp Pro Asn Asp Thr Val Pr - #o Val Phe Tyr Gly Asp     #           110     - Ile Lys Val Gln Ile Leu Asn Asp Ser His Ty - #r Ala Asp Trp Val Met     #       125     - Phe Met Leu Cys Arg Gly Ser Glu Gln Arg Il - #e Leu Arg His Phe His     #   140     - Phe Thr Thr Trp Pro Asp Phe Gly Val Pro As - #n Pro Pro Gln Thr Leu     145                 1 - #50                 1 - #55                 1 -     #60     - Val Arg Phe Val Arg Ala Phe Arg Asp Arg Il - #e Cys Ala Glu Gln Arg     #               175     - Pro Ile Val Val His Cys Ser Ala Gly Val Gl - #y Arg Ser Gly Thr Phe     #           190     - Ile Thr Leu Asp Arg Ile Leu Gln Gln Ile As - #n Thr Ser Asp Tyr Val     #       205     - Asp Ile Phe Gly Ile Val Tyr Ala Met Arg Ly - #s Glu Arg Val Trp Met     #   220     - Val Gln Thr Glu Gln Gln Tyr Ile Cys Ile Hi - #s Gln Cys Leu Leu Ala     225                 2 - #30                 2 - #35                 2 -     #40     - Val Leu Glu Gly Lys Glu Asn Ile Val Gly Pr - #o     #               250     - (2) INFORMATION FOR SEQ ID NO:16:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 255 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:     - Ser Gln Ser Gln Met Val Ala Ser Ala Ser Gl - #u Asn Asn Ala Lys Asn     #                15     - Arg Tyr Arg Asn Val Leu Pro Tyr Asp Trp Se - #r Arg Val Pro Leu Lys     #            30     - Pro Ile His Glu Glu Pro Gly Ser Asp Tyr Il - #e Asn Ala Ser Phe Met     #        45     - Pro Gly Leu Trp Ser Pro Gln Glu Phe Ile Al - #a Thr Gln Gly Pro Leu     #    60     - Pro Gln Thr Val Gly Asp Phe Trp Arg Leu Va - #l Trp Glu Gln Gln Ser     #80     - His Thr Leu Val Met Leu Thr Asn Cys Met Gl - #u Ala Gly Arg Val Lys     #                95     - Cys Glu His Tyr Trp Pro Leu Asp Ser Gln Pr - #o Cys Thr His Gly His     #           110     - Leu Arg Val Thr Leu Val Gly Glu Glu Val Me - #t Glu Asn Trp Thr Val     #       125     - Leu Leu Leu Leu Gln Val Glu Glu Gln Lys Th - #r Leu Ser Val Arg Gln     #   140     - Phe His Tyr Gln Ala Trp Pro Asp His Gly Va - #l Pro Ser Ser Pro Asp     145                 1 - #50                 1 - #55                 1 -     #60     - Thr Leu Leu Ala Phe Trp Arg Met Leu Arg Gl - #n Trp Leu Asp Gln Thr     #               175     - Met Glu Gly Gly Pro Pro Ile Val His Cys Se - #r Ala Gly Val Gly Arg     #           190     - Thr Gly Thr Leu Ile Ala Leu Asp Val Leu Le - #u Arg Gln Leu Gln Ser     #       205     - Glu Gly Leu Leu Gly Pro Phe Ser Phe Val Ar - #g Lys Met Arg Glu Ser     #   220     - Arg Pro Leu Met Val Gln Thr Glu Ala Gln Ty - #r Val Phe Leu His Gln     225                 2 - #30                 2 - #35                 2 -     #40     - Cys Ile Cys Gly Ser Ser Asn Ser Gln Pro Ar - #g Pro Gln Pro Arg     #               255     - (2) INFORMATION FOR SEQ ID NO:17:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 243 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:     - Phe Val Asp Pro Lys Glu Tyr Asp Ile Pro Gl - #y Leu Val Arg Lys Asn     #                15     - Arg Tyr Lys Thr Ile Leu Pro Asn Pro His Se - #r Arg Val Arg Leu Thr     #            30     - Ser Pro Asp Pro Glu Asp Pro Leu Ser Ser Ty - #r Ile Asn Ala Asn Tyr     #        45     - Ile Arg Gly Tyr Asn Gly Glu Glu Lys Val Ty - #r Ile Ala Thr Gln Gly     #    60     - Pro Ile Val Ser Thr Val Val Asp Phe Trp Ar - #g Met Val Trp Gln Glu     #80     - Arg Thr Pro Ile Ile Val Met Ile Thr Asn Il - #e Glu Glu Met Asn Glu     #                95     - Lys Cys Thr Glu Tyr Trp Pro Glu Glu Gln Va - #l Val His Asp Gly Val     #           110     - Glu Ile Thr Val Gln Lys Val Ile His Thr Gl - #u Asp Tyr Arg Leu Ile     #       125     - Ser Leu Arg Arg Gly Thr Glu Glu Arg Gly Le - #u Lys His Tyr Trp Phe     #   140     - Thr Ser Trp Pro Asp Gln Lys Thr Pro Asp Ar - #g Ala Pro Pro Leu Leu     145                 1 - #50                 1 - #55                 1 -     #60     - His Leu Val Arg Glu Val Glu Glu Ala Ala Gl - #n Gln Glu Gly Pro His     #               175     - Cys Ser Pro Ile Ile Val His Cys Ser Ala Gl - #y Ile Gly Arg Thr Gly     #           190     - Cys Phe Ile Ala Thr Ser Ile Cys Cys Gln Gl - #n Leu Arg Arg Glu Gly     #       205     - Val Val Asp Ile Leu Lys Thr Thr Cys Gln Le - #u Arg Gln Asp Arg Gly     #   220     - Gly Met Ile Gln Thr Cys Glu Gln Tyr Gln Ph - #e Val His His Ala Met     225                 2 - #30                 2 - #35                 2 -     #40     - Ser Leu Tyr     - (2) INFORMATION FOR SEQ ID NO:18:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 257 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:     - Asp Arg Thr Thr Lys Asn Ser Asp Leu Lys Gl - #u Asn Ala Cys Lys Asn     #                15     - Arg Tyr Pro Asp Ile Lys Ala Tyr Asp Gln Th - #r Arg Val Lys Leu Ala     #            30     - Val Ile Asn Gly Leu Gln Thr Thr Asp Tyr Il - #e Asn Ala Asn Phe Val     #        45     - Ile Gly Tyr Lys Glu Arg Lys Lys Phe Ile Cy - #s Ala Gln Gly Pro Met     #    60     - Glu Ser Thr Ile Asp Asp Phe Trp Arg Met Il - #e Trp Glu Gln His Leu     #80     - Glu Ile Ile Val Ile Leu Thr Asn Leu Glu Gl - #u Tyr Asn Lys Ala Lys     #                95     - Cys Ala Lys Tyr Trp Pro Glu Lys Val Phe As - #p Thr Lys Gln Phe Gly     #           110     - Asp Ile Leu Val Lys Phe Ala Gln Glu Arg Ly - #s Thr Gly Asp Tyr Ile     #       125     - Glu Leu Asn Val Ser Lys Asn Lys Ala Asn Va - #l Gly Glu Glu Glu Asp     #   140     - Arg Arg Gln Ile Thr Gln Tyr His Tyr Leu Th - #r Trp Lys Asp Phe Met     145                 1 - #50                 1 - #55                 1 -     #60     - Ala Pro Glu His Pro His Gly Ile Ile Lys Ph - #e Ile Arg Gln Ile Asn     #               175     - Ser Val Tyr Ser Leu Gln Arg Gly Pro Ile Le - #u Val His Cys Ser Ala     #           190     - Gly Val Gly Arg Thr Gly Thr Leu Val Ala Le - #u Asp Ser Leu Ile Gln     #       205     - Gln Leu Glu Glu Glu Asp Ser Val Ser Ile Ty - #r Asn Thr Val Cys Asp     #   220     - Leu Arg His Gln Arg Asn Phe Leu Val Gln Se - #r Leu Lys Gln Tyr Ile     225                 2 - #30                 2 - #35                 2 -     #40     - Phe Leu Tyr Arg Ala Leu Leu Asp Thr Gly Th - #r Phe Gly Asn Thr Asp     #               255     - Ile     - (2) INFORMATION FOR SEQ ID NO:19:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 258 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:     - Val Gly Thr Phe His Cys Ser Met Ser Pro Gl - #y Asn Leu Glu Lys Asn     #                15     - Arg Tyr Gly Asp Val Pro Cys Leu Asp Gln Th - #r Arg Val Lys Leu Thr     #            30     - Lys Arg Ser Gly His Thr Gln Thr Asp Tyr Il - #e Asn Ala Ser Phe Met     #        45     - Asp Gly Tyr Lys Gln Lys Asn Ala Tyr Ile Gl - #y Thr Gln Gly Pro Leu     #    60     - Glu Asn Thr Tyr Arg Asp Phe Trp Leu Met Va - #l Trp Glu Gln Lys Val     #80     - Leu Val Ile Val Met Thr Thr Arg Phe Glu Gl - #u Gly Gly Arg Arg Lys     #                95     - Cys Gly Gln Tyr Trp Pro Leu Glu Lys Asp Se - #r Arg Ile Arg Phe Gly     #           110     - Phe Leu Thr Val Thr Asn Leu Gly Val Glu As - #n Met Asn His Tyr Lys     #       125     - Lys Leu Glu Ile His Asn Thr Glu Glu Arg Gl - #n Lys Arg Gln Val Thr     #   140     - His Phe Gln Phe Leu Ser Trp Pro Asp Tyr Gl - #y Val Pro Ser Ser Ala     145                 1 - #50                 1 - #55                 1 -     #60     - Ala Ser Leu Ile Asp Phe Leu Arg Val Val Ar - #g Asn Gln Gln Ser Leu     #               175     - Ala Val Ser Asn Met Gly Ala Arg Ser Lys Gl - #y Gln Cys Pro Glu Pro     #           190     - Pro Ile Val Val His Cys Ser Ala Gly Ile Gl - #y Arg Thr Gly Thr Phe     #       205     - Cys Ser Leu Asp Ile Cys Leu Ala Gln Leu Gl - #u Glu Leu Gly Thr Leu     #   220     - Asn Val Phe Gln Thr Val Ser Arg Met Arg Th - #r Gln Arg Ala Phe Ser     225                 2 - #30                 2 - #35                 2 -     #40     - Ile Gln Thr Pro Glu Gln Tyr Tyr Phe Cys Ty - #r Lys Ala Ile Leu Glu     #               255     - Phe Ala     - (2) INFORMATION FOR SEQ ID NO:20:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 253 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:     - Ile Tyr Pro Thr Ala Thr Gly Glu Lys Glu Gl - #u Asn Val Lys Lys Asn     #                15     - Arg Tyr Lys Asp Ile Leu Pro Phe Asp His Se - #r Arg Val Lys Leu Thr     #            30     - Leu Lys Thr Pro Ser Gln Asp Ser Asp Tyr Il - #e Asn Ala Asn Phe Ile     #        45     - Lys Gly Val Tyr Gly Pro Lys Ala Tyr Val Al - #a Thr Gln Gly Pro Leu     #    60     - Ala Asn Thr Val Ile Asp Phe Trp Arg Met Va - #l Trp Glu Tyr Asn Val     #80     - Val Ile Ile Val Met Ala Cys Arg Glu Phe Gl - #u Met Gly Arg Lys Lys     #                95     - Cys Glu Arg Tyr Trp Pro Leu Tyr Gly Glu As - #p Pro Ile Thr Phe Ala     #           110     - Pro Phe Lys Ile Ser Cys Glu Asp Glu Gln Al - #a Arg Thr Asp Tyr Phe     #       125     - Ile Leu Leu Leu Glu Phe Gln Asn Glu Ser Ar - #g Arg Leu Tyr Gln Phe     #   140     - His Tyr Val Asn Trp Pro Asp His Asp Val Pr - #o Ser Ser Phe Asp Ser     145                 1 - #50                 1 - #55                 1 -     #60     - Ile Leu Asp Met Ile Ser Leu Met Arg Lys Ty - #r Gln Glu His Glu Asp     #               175     - Val Pro Ile Cys Ile His Cys Ser Ala Gly Cy - #s Gly Arg Thr Gly Ala     #           190     - Ile Cys Ala Ile Asp Tyr Thr Trp Asn Leu Le - #u Lys Ala Gly Lys Ile     #       205     - Pro Glu Glu Phe Asn Val Phe Asn Leu Ile Gl - #n Glu Met Arg Thr Gln     #   220     - Arg His Ser Ala Val Gln Thr Lys Glu Gln Ty - #r Glu Leu Val His Arg     225                 2 - #30                 2 - #35                 2 -     #40     - Ala Ile Ala Gln Leu Phe Glu Lys Gln Leu Gl - #n Leu Tyr     #               250     - (2) INFORMATION FOR SEQ ID NO:21:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 242 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:     - Gly Leu Ala Ile Thr Phe Ala Lys Leu Pro Gl - #n Asn Leu Asp Lys Asn     #                15     - Arg Tyr Lys Asp Val Leu Pro Tyr Asp Thr Th - #r Arg Val Leu Leu Gln     #            30     - Gly Asn Glu Asp Tyr Ile Asn Ala Ser Tyr Va - #l Asn Met Glu Ile Pro     #        45     - Ala Ala Asn Leu Val Asn Lys Tyr Ile Ala Th - #r Gln Gly Pro Leu Pro     #    60     - His Thr Cys Ala Gln Phe Trp Gln Val Val Tr - #p Asp Gln Lys Leu Ser     #80     - Leu Ile Val Met Leu Thr Thr Leu Thr Glu Ar - #g Gly Arg Thr Lys Cys     #                95     - His Gln Tyr Trp Pro Asp Pro Pro Asp Val Me - #t Asn His Gly Gly Phe     #           110     - His Ile Gln Cys Gln Ser Glu Asp Cys Thr Il - #e Ala Tyr Val Ser Met     #       125     - Leu Val Thr Asn Thr Gln Thr Gly Glu Glu Hi - #s Thr Val Thr His Leu     #   140     - Gln Tyr Val Ala Trp Pro Asp His Gly Ile Pr - #o Asp Asp Ser Ser Asp     145                 1 - #50                 1 - #55                 1 -     #60     - Phe Leu Glu Phe Val Asn Tyr Val Arg Ser Le - #u Arg Val Asp Ser Glu     #               175     - Pro Val Leu Val His Cys Ser Ala Gly Ile Gl - #y Arg Thr Gly Val Leu     #           190     - Val Thr Met Glu Thr Ala Met Cys Leu Thr Gl - #u Arg Asn Leu Pro Ile     #       205     - Tyr Pro Leu Asp Ile Val Arg Lys Met Arg As - #p Gln Arg Ala Met Met     #   220     - Val Gln Thr Ser Ser Gln Tyr Lys Phe Val Cy - #s Glu Ala Ile Leu Arg     225                 2 - #30                 2 - #35                 2 -     #40     - Val Tyr     - (2) INFORMATION FOR SEQ ID NO:22:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 277 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:     - Pro Ser Glu Thr Ser Glu Gly Asp Lys Lys Hi - #s Asn Thr Ser Lys Asn     #                15     - Arg Tyr Thr Asn Ile Leu Pro Val Asn His Th - #r Arg Val Gln Leu Lys     #            30     - Lys Ile Gln Asp Lys Glu Gly Ser Asp Tyr Il - #e Asn Ala Asn Tyr Ile     #        45     - Asp Gly Ala Tyr Pro Lys Gln Phe Ile Cys Th - #r Gln Gly Pro Leu Pro     #    60     - Asn Thr Ile Ala Asp Phe Trp Arg Met Val Tr - #p Glu Asn Arg Cys Arg     #80     - Ile Ile Val Met Leu Ser Arg Glu Ser Glu Gl - #y Ser Glu Asn Cys Arg     #                95     - Ile Lys Cys Asp Arg Tyr Trp Pro Glu Gln Il - #e Gly Gly Glu Gln Phe     #           110     - Ser Ile Tyr Gly Asn Gly Asn Glu Val Phe Gl - #y Thr Tyr Ser Val Glu     #       125     - Leu Val Glu Val Ile Gln Cys Arg Glu Ile Il - #e Thr Arg Asn Ile Arg     #   140     - Leu Thr Phe Glu Gly Glu Thr Arg Asp Ile Th - #r Gln Tyr Gln Tyr Glu     145                 1 - #50                 1 - #55                 1 -     #60     - Gly Trp Pro Asp His Asn Ile Pro Asp His Th - #r Gln Pro Phe Arg Gln     #               175     - Leu Leu His Ser Ile Thr Asn Arg Gln Asn Gl - #n Ile Ile Pro Ser Ser     #           190     - Asp Arg Asn Val Pro Ile Ile Val His Cys Se - #r Ala Gly Val Gly Arg     #       205     - Thr Gly Thr Phe Cys Thr Ala Val Ile Met Me - #t Lys Lys Leu Asp His     #   220     - Tyr Phe Lys Gln Leu Asp Tyr Asn Ser Arg Il - #e Asp Phe Asn Leu Phe     225                 2 - #30                 2 - #35                 2 -     #40     - Ser Ile Val Leu Lys Leu Arg Glu Gln Arg Pr - #o Gly Met Val Gln Gln     #               255     - Leu Glu Gln Tyr Leu Phe Cys Tyr Lys Thr Il - #e Leu Asp Glu Ile Tyr     #           270     - His Arg Leu Asn Cys             275     - (2) INFORMATION FOR SEQ ID NO:23:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 254 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:     - Gln Trp Ser Thr Val Asp Ser Leu Ser Asn Th - #r Ser Tyr Lys Lys Asn     #                15     - Arg Tyr Thr Asp Ile Val Pro Tyr Asn Cys Th - #r Arg Val His Leu Lys     #            30     - Arg Thr Ser Pro Ser Glu Leu Asp Tyr Ile As - #n Ala Ser Phe Ile Lys     #        45     - Thr Glu Thr Ser Asn Tyr Ile Ala Cys Gln Gl - #y Ser Ile Ser Arg Ser     #    60     - Ile Ser Asp Phe Trp His Met Val Trp Asp As - #n Val Glu Asn Ile Gly     #80     - Thr Ile Val Met Leu Gly Ser Leu Phe Glu Al - #a Gly Arg Glu Met Cys     #                95     - Thr Ala Tyr Trp Pro Ser Asn Gly Ile Gly As - #p Lys Gln Val Tyr Gly     #           110     - Asp Tyr Cys Val Lys Gln Ile Ser Glu Glu As - #n Val Asp Asn Ser Arg     #       125     - Phe Ile Leu Phe Glu Ile Gln Asn Ala Asn Ph - #e Pro Ser Val Lys Lys     #   140     - Val His His Tyr Gln Tyr Pro Asn Trp Ser As - #p Cys Asn Ser Pro Glu     145                 1 - #50                 1 - #55                 1 -     #60     - Asn Val Lys Ser Met Val Glu Phe Leu Lys Ty - #r Val Asn Asn Ser His     #               175     - Gly Ser Gly Asn Thr Ile Val His Cys Ser Al - #a Gly Val Gly Arg Thr     #           190     - Gly Thr Phe Ile Val Leu Asp Thr Ile Leu Ar - #g Phe Pro Glu Ser Lys     #       205     - Leu Ser Gly Phe Asn Pro Ser Val Ala Asp Se - #r Ser Asp Val Val Phe     #   220     - Gln Leu Val Asp His Ile Arg Lys Gln Arg Me - #t Lys Met Val Gln Thr     225                 2 - #30                 2 - #35                 2 -     #40     - Phe Thr Gln Phe Lys Tyr Val Tyr Asp Leu Il - #e Asp Ser Leu     #               250     - (2) INFORMATION FOR SEQ ID NO:24:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 250 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:     - Trp Cys Cys Leu Ala Ser Ser Arg Ser Thr Se - #r Ile Ser Arg Lys Asn     #                15     - Arg Tyr Thr Asp Ile Val Pro Tyr Asp Lys Th - #r Arg Val Arg Leu Ala     #            30     - Val Pro Lys Gly Cys Ser Asp Tyr Ile Asn Al - #a Ser His Ile Asp Val     #        45     - Gly Asn Lys Lys Tyr Ile Ala Cys Gln Ala Pr - #o Lys Pro Gly Thr Leu     #    60     - Leu Asp Phe Trp Glu Met Val Trp His Asn Se - #r Gly Thr Asn Gly Val     #80     - Ile Val Met Leu Thr Asn Leu Tyr Glu Ala Gl - #y Ser Glu Lys Cys Ser     #                95     - Gln Tyr Trp Pro Asp Asn Lys Asp His Ala Le - #u Cys Leu Glu Gly Gly     #           110     - Leu Arg Ile Ser Val Gln Lys Tyr Glu Thr Ph - #e Glu Asp Leu Lys Val     #       125     - His Leu Phe Arg Leu Asp Lys Pro Asn Gly Pr - #o Pro Lys Tyr Ile His     #   140     - His Phe Trp Val His Thr Trp Phe Asp Lys Th - #r His Pro Asp Ile Glu     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Ile Thr Gly Leu Ile Arg Cys Ile Asp Ly - #s Val Pro Asn Asp Gly     #               175     - Pro Met Phe Val His Cys Ser Ala Gly Val Gl - #y Arg Thr Gly Thr Phe     #           190     - Ile Ala Val Asp Gln Ile Leu Gln Val Pro Ly - #s Asn Ile Leu Pro Lys     #       205     - Thr Thr Asn Leu Glu Asp Ser Lys Asp Phe Il - #e Phe Asn Cys Val Asn     #   220     - Ser Leu Arg Ser Gln Arg Met Lys Met Val Gl - #n Asn Phe Glu Gln Phe     225                 2 - #30                 2 - #35                 2 -     #40     - Lys Phe Leu Tyr Asp Val Val Asp Tyr Leu     #               250     - (2) INFORMATION FOR SEQ ID NO:25:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 260 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:     - Gly Ile Thr Ala Asp Ser Ser Asn His Pro As - #p Asn Lys His Lys Asn     #                15     - Arg Tyr Ile Asn Ile Val Ala Tyr Asp His Se - #r Arg Val Lys Leu Ala     #            30     - Gln Leu Ala Glu Lys Asp Gly Lys Leu Thr As - #p Tyr Ile Asn Ala Asn     #        45     - Tyr Val Asp Gly Tyr Asn Arg Pro Lys Ala Ty - #r Ile Ala Ala Gln Gly     #    60     - Pro Leu Lys Ser Thr Ala Glu Asp Phe Trp Ar - #g Met Ile Trp Glu His     #80     - Asn Val Glu Val Ile Val Met Ile Thr Asn Le - #u Val Glu Lys Gly Arg     #                95     - Arg Lys Cys Asp Gln Tyr Trp Pro Ala Asp Gl - #y Ser Glu Glu Tyr Gly     #           110     - Asn Phe Leu Val Thr Gln Lys Ser Val Gln Va - #l Leu Ala Tyr Tyr Thr     #       125     - Val Phe Thr Leu Arg Asn Thr Lys Ile Lys Ly - #s Gly Ser Gln Lys Gly     #   140     - Arg Pro Ser Gly Arg Val Val Thr Gln Tyr Hi - #s Tyr Thr Gln Trp Pro     145                 1 - #50                 1 - #55                 1 -     #60     - Asp Met Gly Val Pro Glu Tyr Ser Leu Pro Va - #l Leu Thr Phe Val Arg     #               175     - Lys Ala Ala Tyr Ala Lys Arg His Ala Val Gl - #y Pro Val Val Val His     #           190     - Cys Ser Ala Gly Val Gly Arg Thr Gly Thr Ty - #r Ile Val Leu Asp Ser     #       205     - Met Leu Gln Gln Ile Gln His Glu Gly Thr Va - #l Asn Ile Phe Gly Phe     #   220     - Leu Lys His Ile Arg Ser Gln Arg Asn Tyr Le - #u Val Gln Thr Glu Glu     225                 2 - #30                 2 - #35                 2 -     #40     - Gln Tyr Val Phe Ile His Asp Thr Leu Val Gl - #u Ala Ile Leu Ser Lys     #               255     - Glu Thr Glu Val                 260     - (2) INFORMATION FOR SEQ ID NO:26:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 245 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:     - Thr Ser Arg Phe Ile Ser Ala Asn Leu Pro Cy - #s Asn Lys Phe Lys Asn     #                15     - Arg Leu Val Asn Ile Met Pro Tyr Glu Leu Th - #r Arg Val Cys Leu Gln     #            30     - Pro Ile Arg Gly Val Glu Gly Ser Asp Tyr Il - #e Asn Ala Ser Phe Leu     #        45     - Asp Gly Tyr Arg Gln Gln Lys Ala Tyr Ile Al - #a Thr Gln Gly Pro Leu     #    60     - Ala Glu Ser Thr Glu Asp Phe Trp Arg Met Le - #u Trp Glu His Asn Ser     #80     - Thr Ile Ile Val Met Leu Thr Lys Leu Arg Gl - #u Met Gly Arg Glu Lys     #                95     - Cys His Gln Tyr Trp Pro Ala Glu Arg Ser Al - #a Arg Tyr Gln Tyr Phe     #           110     - Val Val Asp Pro Met Ala Glu Tyr Asn Met Pr - #o Gln Tyr Ile Leu Phe     #       125     - Lys Val Thr Asp Ala Arg Asp Gly Gln Ser Ar - #g Thr Ile Arg Gln Phe     #   140     - Gln Phe Thr Asp Trp Pro Glu Gln Gly Val Pr - #o Lys Thr Gly Glu Gly     145                 1 - #50                 1 - #55                 1 -     #60     - Phe Ile Asp Phe Ile Gly Gln Val His Lys Th - #r Lys Glu Gln Phe Gly     #               175     - Gln Asp Gly Pro Ile Thr Val His Cys Ser Al - #a Gly Val Gly Arg Thr     #           190     - Gly Val Phe Ile Thr Leu Ser Ile Val Leu Gl - #u Arg Met Arg Tyr Glu     #       205     - Gly Val Val Asp Met Phe Gln Thr Val Lys Th - #r Leu Arg Thr Gln Arg     #   220     - Pro Ala Met Val Gln Thr Glu Asp Gln Tyr Gl - #n Leu Cys Tyr Arg Ala     225                 2 - #30                 2 - #35                 2 -     #40     - Ala Leu Glu Tyr Leu                     245     - (2) INFORMATION FOR SEQ ID NO:27:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 232 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:     - Asn Asp Lys Met Arg Thr Gly Asn Leu Pro Al - #a Asn Met Lys Lys Asn     #                15     - Arg Val Leu Gln Ile Ile Pro Tyr Glu Phe As - #n Arg Val Ile Ile Pro     #            30     - Val Lys Arg Gly Glu Asn Asp Lys Met Arg Th - #r Gly Asn Leu Pro Ala     #        45     - Asn Met Lys Lys Asn Arg Val Leu Gln Ile Il - #e Pro Tyr Glu Phe Asn     #    60     - Arg Val Ile Ile Pro Val Lys Arg Gly Glu Gl - #u Asn Thr Asp Tyr Val     #80     - Asn Ala Ser Phe Ile Asp Gly Tyr Arg Gln Ly - #s Asp Ser Tyr Ile Ala     #                95     - Ser Gln Gly Pro Leu Leu His Thr Ile Glu As - #p Phe Trp Arg Met Ile     #           110     - Trp Glu Trp Lys Ser Cys Ser Ile Val Met Le - #u Thr Glu Leu Glu Glu     #       125     - Arg Gly Gln Glu Lys Cys Ala Gln Tyr Trp Pr - #o Ser Asp Gly Leu Val     #   140     - Ser Tyr Gly Asp Ile Thr Val Glu Leu Lys Ly - #s Glu Glu Glu Cys Glu     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Tyr Thr Val Leu Leu Val Thr Asn Thr Ar - #g Glu Asn Lys Ser Arg     #               175     - Gln Ile Arg Gln Phe His Phe His Gly Trp Pr - #o Glu Val Gly Ile Pro     #           190     - Ser Asp Gly Lys Gly Met Ile Ser Ile Ile Al - #a Ala Val Gln Lys Gln     #       205     - Gln Gln Gln Ser Gly Asn His Pro Ile Thr Va - #l His Cys Ser Ala Gly     #   220     - Ala Gly Arg Thr Gly Thr Phe Cys     225                 2 - #30     - (2) INFORMATION FOR SEQ ID NO:28:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 249 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:     - Lys Glu Asn Met Arg Thr Gly Asn Leu Pro Al - #a Asn Met Lys Lys Ala     #                15     - Arg Val Ile Gln Ile Ile Pro Tyr Asp Phe As - #n Arg Val Ile Leu Ser     #            30     - Met Lys Arg Gly Gln Glu Tyr Thr Asp Tyr Il - #e Asn Ala Ser Phe Ile     #        45     - Asp Gly Tyr Arg Gln Lys Asp Tyr Phe Ile Al - #a Thr Gln Gly Pro Leu     #    60     - Ala His Thr Val Glu Asp Phe Trp Arg Met Il - #e Trp Glu Trp Lys Ser     #80     - His Thr Ile Val Met Leu Thr Glu Val Gln Gl - #u Arg Glu Gln Asp Lys     #                95     - Cys Tyr Gln Tyr Trp Pro Thr Glu Gly Ser Va - #l Thr His Gly Glu Ile     #           110     - Thr Ile Glu Ile Lys Asn Asp Thr Leu Ser Gl - #u Ala Ile Ser Ile Phe     #       125     - Leu Val Thr Leu Asn Gln Pro Gln Ala Arg Gl - #n Glu Glu Gln Val Arg     #   140     - Val Val Arg Gln Phe His Phe His Gly Trp Pr - #o Glu Ile Gly Ile Pro     145                 1 - #50                 1 - #55                 1 -     #60     - Ala Glu Gly Lys Gly Met Ile Asp Leu Ile Al - #a Ala Val Gln Lys Gln     #               175     - Gln Gln Gln Thr Gly Asn His Pro Ile Thr Va - #l His Cys Ser Ala Gly     #           190     - Ala Gly Arg Thr Gly Thr Phe Ile Ala Leu Se - #r Asn Ile Leu Glu Arg     #       205     - Val Lys Ala Glu Gly Leu Leu Asp Val Phe Gl - #n Ala Val Lys Ser Leu     #   220     - Arg Leu Gln Arg Pro His Met Val Gln Thr Le - #u Glu Gln Tyr Glu Phe     225                 2 - #30                 2 - #35                 2 -     #40     - Cys Tyr Lys Val Val Gln Asp Phe Ile                     245     - (2) INFORMATION FOR SEQ ID NO:29:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 249 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:     - Val Glu Asp Cys Ser Ile Ala Leu Leu Pro Ar - #g Asn His Glu Lys Asn     #                15     - Arg Cys Met Asp Ile Leu Pro Pro Asp Arg Cy - #s Leu Pro Phe Leu Ile     #            30     - Thr Ile Asp Gly Glu Ser Ser Asn Tyr Ile As - #n Ala Ala Leu Met Asp     #        45     - Ser Tyr Lys Gln Pro Ser Ala Phe Ile Val Th - #r Gln His Pro Leu Pro     #    60     - Asn Thr Val Lys Asp Phe Trp Arg Leu Val Le - #u Asp Tyr His Cys Thr     #80     - Ser Val Val Met Leu Asn Asp Val Asp Pro Al - #a Gln Leu Cys Pro Gln     #                95     - Tyr Trp Pro Glu Asn Gly Val His Arg His Gl - #y Pro Ile Gln Val Glu     #           110     - Phe Val Ser Ala Asp Leu Glu Glu Asp Ile Il - #e Ser Phe Arg Ile Tyr     #       125     - Asn Ala Ala Arg Pro Gln Asp Gly Tyr Arg Me - #t Val Gln Gln Phe Gln     #   140     - Phe Leu Gly Trp Pro Met Tyr Arg Asp Thr Pr - #o Val Ser Lys Arg Ser     145                 1 - #50                 1 - #55                 1 -     #60     - Phe Leu Lys Leu Ile Arg Gln Val Asp Lys Tr - #p Gln Glu Glu Tyr Asn     #               175     - Gly Gly Glu Gly Pro Thr Val Val His Cys Le - #u Asn Gly Gly Gly Arg     #           190     - Ser Gly Thr Phe Cys Ala Ile Ser Ile Val Cy - #s Glu Met Leu Arg His     #       205     - Gln Arg Thr Val Asp Val Phe His Ala Val Ly - #s Thr Leu Arg Asn Asn     #   220     - Lys Pro Asn Met Val Asp Leu Leu Asp Gln Ty - #r Lys Phe Cys Tyr Glu     225                 2 - #30                 2 - #35                 2 -     #40     - Val Ala Leu Glu Tyr Leu Asn Ser Gly                     245     - (2) INFORMATION FOR SEQ ID NO:30:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 277 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:     - Trp Arg Thr Gln His Ile Gly Asn Gln Glu Gl - #u Asn Lys Lys Lys Asn     #                15     - Arg Asn Ser Asn Val Val Pro Tyr Asp Phe As - #n Arg Val Pro Leu Lys     #            30     - His Glu Leu Glu Met Ser Lys Glu Ser Glu Pr - #o Glu Ser Asp Glu Ser     #        45     - Ser Asp Asp Asp Ser Asp Ser Glu Glu Thr Se - #r Lys Tyr Ile Asn Ala     #    60     - Ser Phe Val Met Ser Tyr Trp Lys Pro Glu Me - #t Met Ile Ala Ala Gln     #80     - Gly Pro Leu Lys Glu Thr Ile Gly Asp Phe Tr - #p Gln Met Ile Phe Gln     #                95     - Arg Lys Val Lys Val Ile Val Met Leu Thr Gl - #u Leu Val Asn Gly Asp     #           110     - Gln Glu Val Cys Ala Gln Tyr Trp Gly Glu Gl - #y Lys Gln Thr Tyr Gly     #       125     - Asp Met Glu Val Glu Met Lys Asp Thr Asn Ar - #g Ala Ser Ala Tyr Thr     #   140     - Leu Phe Glu Leu Arg His Ser Lys Arg Lys Gl - #u Pro Arg Thr Val Tyr     145                 1 - #50                 1 - #55                 1 -     #60     - Gln Tyr Gln Cys Thr Thr Trp Lys Gly Glu Gl - #u Leu Pro Ala Glu Pro     #               175     - Lys Asp Leu Val Ser Met Ile Gln Asp Leu Ly - #s Gln Lys Leu Pro Lys     #           190     - Ala Ser Pro Glu Gly Met Lys Tyr His Lys Hi - #s Ala Ser Ile Leu Val     #       205     - His Cys Arg Asp Gly Ser Gln Gln Thr Gly Le - #u Phe Cys Ala Leu Phe     #   220     - Asn Leu Leu Glu Ser Ala Glu Thr Glu Asp Va - #l Val Asp Val Phe Gln     225                 2 - #30                 2 - #35                 2 -     #40     - Val Val Lys Ser Leu Arg Lys Ala Arg Pro Gl - #y Val Val Cys Ser Tyr     #               255     - Glu Gln Tyr Gln Phe Leu Tyr Asp Ile Ile Al - #a Ser Ile Tyr Pro Ala     #           270     - Gln Asn Gly Gln Val             275     - (2) INFORMATION FOR SEQ ID NO:31:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 247 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:     - Ser Lys Ser Cys Ser Val Gly Glu Asn Glu Gl - #u Asn Asn Met Lys Asn     #                15     - Arg Ser Gln Glu Ile Ile Pro Tyr Asp Arg As - #n Arg Val Ile Leu Thr     #            30     - Pro Leu Pro Met Arg Glu Asn Ser Thr Tyr Il - #e Asn Ala Ser Phe Ile     #        45     - Glu Gly Tyr Asp Asn Ser Glu Thr Phe Ile Il - #e Ala Gln Asp Pro Phe     #    60     - Glu Asn Thr Ile Gly Asp Phe Trp Arg Met Il - #e Ser Glu Gln Ser Val     #80     - Thr Thr Leu Val Met Ile Ser Glu Ile Gly As - #p Gly Pro Arg Lys Cys     #                95     - Pro Arg Tyr Trp Ala Asp Asp Glu Val Gln Ty - #r Asp His Ile Leu Val     #           110     - Lys Tyr Val His Ser Glu Ser Cys Pro Tyr Ty - #r Thr Phe Phe Tyr Val     #       125     - Thr Asn Cys Lys Ile Asp Asp Thr Leu Lys Va - #l Thr Gln Phe Gln Tyr     #   140     - Asn Gly Trp Pro Thr Val Asp Gly Glu Val Pr - #o Glu Val Cys Arg Gly     145                 1 - #50                 1 - #55                 1 -     #60     - Ile Ile Glu Leu Val Asp Gln Ala Tyr Asn Hi - #s Tyr Lys Asn Asn Lys     #               175     - Asn Ser Gly Cys Arg Ser Pro Leu Thr Val Hi - #s Cys Ser Leu Gly Thr     #           190     - Asp Arg Ser Ser Ile Phe Val Ala Met Cys Il - #e Leu Val Gln His Leu     #       205     - Arg Leu Glu Lys Cys Val Asp Ile Cys Ala Th - #r Thr Arg Lys Leu Arg     #   220     - Ser Gln Arg Thr Gly Leu Ile Asn Ser Tyr Al - #a Gln Tyr Glu Phe Leu     225                 2 - #30                 2 - #35                 2 -     #40     - His Arg Ala Ile Ile Asn Tyr                     245     - (2) INFORMATION FOR SEQ ID NO:32:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 252 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:     - Gln Ser Asp Tyr Ser Ala Ala Leu Lys Gln Cy - #s Asn Arg Glu Lys Asn     #                15     - Arg Thr Ser Ser Ile Ile Pro Val Glu Arg Se - #r Arg Val Gly Ile Ser     #            30     - Ser Leu Ser Gly Glu Gly Thr Asp Tyr Ile As - #n Ala Ser Tyr Ile Met     #        45     - Gly Tyr Tyr Gln Ser Asn Glu Phe Ile Ile Th - #r Gln His Pro Leu Leu     #    60     - His Thr Ile Lys Asp Phe Trp Arg Met Ile Tr - #p Asp His Asn Ala Gln     #80     - Leu Val Val Met Ile Pro Asp Gly Gln Asn Me - #t Ala Glu Asp Glu Phe     #                95     - Val Tyr Trp Pro Asn Lys Asp Glu Pro Ile As - #n Cys Glu Ser Phe Lys     #           110     - Val Thr Leu Met Ala Glu Glu His Lys Cys Le - #u Ser Asn Glu Glu Lys     #       125     - Leu Ile Ile Phe Ile Leu Glu Ala Thr Gln As - #p Asp Tyr Val Leu Glu     #   140     - Val Arg His Phe Gln Cys Pro Lys Trp Pro As - #n Pro Asp Ser Pro Ile     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Lys Thr Phe Glu Leu Ile Ser Val Ile Ly - #s Glu Glu Ala Ala Asn     #               175     - Arg Asp Gly Pro Met Ile Val His Asp Glu Hi - #s Gly Gly Val Thr Ala     #           190     - Gly Thr Phe Cys Ala Leu Thr Thr Leu Met Hi - #s Gln Leu Glu Lys Glu     #       205     - Asn Ser Val Asp Val Tyr Gln Val Ala Lys Me - #t Ile Asn Leu Met Arg     #   220     - Pro Gly Val Phe Ala Asp Ile Glu Gln Tyr Gl - #n Phe Leu Tyr Lys Val     225                 2 - #30                 2 - #35                 2 -     #40     - Ile Leu Ser Leu Val Ser Thr Arg Gln Glu Gl - #u Asn     #               250     - (2) INFORMATION FOR SEQ ID NO:33:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 252 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:     - Val Glu Cys Phe Ser Ala Gln Lys Glu Cys As - #n Lys Glu Lys Asn Arg     #                15     - Asn Ser Ser Val Val Pro Ser Glu Arg Ala Ar - #g Val Gly Leu Ala Pro     #            30     - Leu Pro Gly Met Lys Gly Thr Asp Tyr Ile As - #n Ala Ser Tyr Ile Met     #        45     - Gly Tyr Tyr Arg Ser Asn Glu Phe Ile Ile Th - #r Gln His Pro Leu Pro     #    60     - His Thr Thr Lys Asp Phe Trp Arg Met Ile Tr - #p Asp His Asn Ala Gln     #80     - Ile Ile Val Met Leu Pro Asp Asn Gln Ser Le - #u Ala Glu Asp Glu Phe     #                95     - Val Tyr Trp Pro Ser Arg Glu Glu Ser Met As - #n Cys Glu Ala Phe Thr     #           110     - Val Thr Leu Ile Ser Lys Asp Arg Leu Cys Le - #u Ser Asn Glu Glu Gln     #       125     - Ile Ile Ile Phe Ile Leu Glu Ala Thr Gln As - #p Asp Tyr Val Leu Glu     #   140     - Val Arg His Phe Gln Cys Pro Lys Trp Pro As - #n Pro Asp Ala Pro Ile     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Ser Thr Phe Glu Leu Ile Asn Val Ile Ly - #s Glu Glu Ala Leu Thr     #               175     - Arg Asp Gly Pro Thr Ile Val His Asp Glu Ty - #r Gly Ala Val Ser Ala     #           190     - Gly Met Leu Cys Ala Leu Thr Thr Leu Ser Gl - #n Gln Leu Glu Asn Glu     #       205     - Asn Ala Val Asp Val Phe Gln Val Ala Lys Me - #t Ile Asn Leu Met Arg     #   220     - Pro Gly Val Phe Thr Asp Ile Glu Gln Tyr Gl - #n Phe Ile Tyr Lys Ala     225                 2 - #30                 2 - #35                 2 -     #40     - Met Leu Ser Leu Val Ser Thr Lys Glu Asn Gl - #y Asn     #               250     - (2) INFORMATION FOR SEQ ID NO:34:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 278 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:     - Glu Thr Asn Leu Met Ala Glu Gln Val Glu Gl - #u Leu Lys Asn Cys Thr     #                15     - Pro Tyr Leu Glu Gln Gln Tyr Lys Asn Ile Il - #e Gln Phe Gln Pro Lys     #            30     - Asp Ile His Ile Ala Ser Ala Met Lys Gln Va - #l Asn Ser Ile Lys Asn     #        45     - Arg Gly Ala Ile Phe Pro Ile Glu Gly Ser Ar - #g Val His Leu Thr Pro     #    60     - Lys Pro Gly Glu Asp Gly Ser Asp Tyr Ile As - #n Ala Ser Trp Leu His     #80     - Gly Phe Arg Arg Leu Arg Asp Phe Ile Val Th - #r Gln His Pro Met Ala     #                95     - His Thr Ile Lys Asp Phe Trp Gln Met Val Tr - #p Asp His Asn Ala Gln     #           110     - Thr Val Val Leu Leu Ser Ser Leu Asp Asp Il - #e Asn Phe Ala Gln Phe     #       125     - Trp Pro Asp Glu Ala Thr Pro Ile Glu Ser As - #p His Tyr Arg Val Lys     #   140     - Phe Leu Asn Lys Thr Asn Lys Ser Asp Tyr Va - #l Ser Phe Val Ile Gln     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Ile Gln Asp Asp Tyr Glu Leu Thr Val Ly - #s Met Leu His Cys Pro     #               175     - Ser Trp Pro Glu Met Ser Asn Pro Asn Ser Il - #e Tyr Asp Phe Ile Val     #           190     - Asp Val His Glu Arg Cys Asn Asp Tyr Arg As - #n Gly Pro Ile Val Ile     #       205     - Val Asp Arg Tyr Gly Gly Ala Gln Ala Cys Th - #r Phe Cys Ala Ile Ser     #   220     - Ser Leu Ala Ile Glu Met Glu Tyr Cys Ser Th - #r Ala Asn Val Tyr Gln     225                 2 - #30                 2 - #35                 2 -     #40     - Tyr Ala Lys Leu Tyr His Asn Lys Arg Pro Gl - #y Val Trp Thr Ser Ser     #               255     - Glu Asp Ile Arg Val Ile Tyr Asn Ile Leu Se - #r Phe Leu Pro Gly Asn     #           270     - Leu Asn Leu Leu Lys Arg             275     - (2) INFORMATION FOR SEQ ID NO:35:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 243 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:     - Thr Asn Asp Pro Arg Tyr Leu Gln Ala Cys Gl - #y Gly Glu Lys Ile Leu     #                15     - Asn Arg Phe Arg Asp Ile Gln Cys Cys Arg Gl - #n Thr Ala Val Arg Ala     #            30     - Asp Asn Tyr Ile Gln Val Gly Asn Thr Arg Th - #r Ile Ala Cys Gln Tyr     #        45     - Pro Leu Gln Ser Gln Leu Glu Ser His Phe Ar - #g Met Leu Ala Glu Asn     #    60     - Arg Thr Pro Val Leu Ala Val Leu Ala Ser Se - #r Ser Glu Ile Ala Asn     #80     - Gln Arg Phe Gly Met Pro Asp Tyr Phe Arg Gl - #n Ser Gly Thr Tyr Gly     #                95     - Ser Ile Thr Val Glu Ser Lys Met Thr Gln Gl - #n Val Gly Leu Gly Asp     #           110     - Gly Ile Asn Met Tyr Thr Leu Thr Ile Arg Gl - #u Ala Gly Gln Lys Thr     #       125     - Ile Ser Val Pro Val Val His Val Gly Asn Tr - #p Pro Asp Gln Thr Ala     #   140     - Val Ser Ser Glu Val Thr Lys Ala Leu Ala Se - #r Leu Val Asp Gln Thr     145                 1 - #50                 1 - #55                 1 -     #60     - Ala Glu Thr Lys Arg Asn Met Tyr Glu Ser Ly - #s Gly Ser Ser Ala Val     #               175     - Ala Asp Asp Ser Lys Leu Arg Pro Val Ile Hi - #s Cys Arg Ala Gly Val     #           190     - Gly Arg Thr Ala Gln Leu Ile Gly Ala Met Cy - #s Met Asn Asp Ser Arg     #       205     - Asn Ser Gln Leu Ser Val Glu Asp Met Val Se - #r Gln Met Arg Val Gln     #   220     - Arg Asn Gly Met Val Gln Lys Asp Glu Gln Le - #u Asp Val Leu Ile Lys     225                 2 - #30                 2 - #35                 2 -     #40     - Leu Ala Glu     - (2) INFORMATION FOR SEQ ID NO:36:     -      (i) SEQUENCE CHARACTERISTICS:     #acids    (A) LENGTH: 11 amino               (B) TYPE: amino acid               (C) STRANDEDNESS: single               (D) TOPOLOGY: linear     -     (ii) MOLECULE TYPE: peptide     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:     - Xaa His Cys Xaa Ala Gly Xaa Xaa Arg Xaa Gl - #y     #                10     __________________________________________________________________________ 

We claim:
 1. A method of reducing the activity of a tyrosine phosphorylated protein, comprising administering to a mammal a protein tyrosine phosphatase in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, whereby formation of the complex reduces the activity of the tyrosine phosphorylated protein.
 2. A method according to claim 1, wherein the tyrosine phosphorylated protein is selected from the group consisting of: p130^(cas), the EGF receptor, p210 bcr:abl, MAP kinase and the insulin receptor.
 3. A method according to claim 1, wherein the protein tyrosine phosphatase is selected from the group consisting of: PTP1B, PTP-PEST, PTP.sub.γ, MKP-1, DEP-1, PTPμ,PTPX1, PTPX10 and PTPH1.
 4. A method of reducing the transforming effects of oncogenes associated with p130^(cas) phosphorylation comprising administering to a mammal a protein tyrosine phosphatase which is PTP-PEST or PTP-PEST in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, whereby the PTP binds to and/or dephosphorylates p130^(cas), thereby negatively regulating the downstream effects of p130^(cas) and reducing the transforming effects of oncogenes associated with p130^(cas) phosphorylation.
 5. A method according to claim 4, wherein the oncogene is selected from the group consisting of: v-crk, v-src and c-Ha-ras.
 6. A method of reducing formation of signalling complexes associated with p130^(cas) comprising administering to a mammal a protein tyrosine phosphatase which is PTP-PEST or PTP-PEST in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, whereby the PTP binds to and/or dephosphorylates p130^(cas), thereby negatively regulating the downstream effects of p130^(cas) and reducing the formation of signalling complexes associated with p130^(cas).
 7. A method according to claim 6, which prevents the induction of mitogenic pathways.
 8. A method of reducing cytotoxic effects associated with protein tyrosine phosphatase administration or overexpression, comprising administering to a mammal a protein tyrosine phosphatase in which the invariant aspartate residue is replaced with an amino acid which does not cause significant alteration of the Km of the enzyme but which results in a reduction in Kcat to less than 1 per minute, in place of a corresponding wild type protein tyrosine phosphatase. 